hypothesis of enzymes

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Introduction to enzymes and their applications

Saurabh Bhatia Published September 2018 • Copyright © IOP Publishing Ltd 2018 Pages 1-1 to 1-29

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Amity institute of Pharmacy, Amity university. Gurgaon, Haryana, India

Published September 2018

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https://doi.org/10.1088/978-0-7503-1302-5ch1

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Enzyme catalysis is an area of fundamental importance in different areas. This chapter offers a concise overview to the fundamental principles and mechanisms of action, catalysis inhibition and its pharmaceutical applications. Additionally, this section also covers basics information related with enzymes such as its structure, function and different properties.

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1.1. Introduction

The cell is the structural and functional unit of life—the basic building block of living systems. Cells have the capability to effectively utilize biocatalysts, known as enzymes, which have outstanding catalytic efficiency and both substrate and reaction specificity. Enzymes have amazing catalytic power and their high level of specificity for their substrate makes them suitable for biological reactions. They are crucial for cellular metabolism. Each and every chemical reaction that takes place in plants, micro-organisms and animals proceeds at a quantifiable rate as a direct result of enzymatic catalysis. Most of the history of biochemistry is directly or indirectly related to the history of enzyme research. Catalysis in biological systems was initially reported in the early 1800s based on research into the digestion of meat. In this report the catalytic activity of secretions from the stomach, the conversion of starch into sugar by saliva, and various plant extracts were reported.

In 1837, Berzelius documented the catalytic nature of fermentation. In the 1850s Louis Pasteur reported that fermentation was a process initiated by living organisms. During this study it was reported that the fermentation of sugar into alcohol by yeast was catalyzed by ferments. He also hypothesized that these ferments are close to the structure of yeast. These ferments were later called enzymes (in yeast). The key breakthrough in the history of enzymes came in 1897 when Edward Buchner isolated, from yeast cells, the soluble active form of the set of enzymes that catalyzes the fermentation of sugar to alcohol. Emul Fischer reported the first systematic studies on enzyme specificity in the early twentieth century [ 1 ]. Later, in 1926, James Sumner extracted urease in pure crystalline form from jack beans [ 2 ]. He also recognized the protein nature of urease . In 1930, John Northrop and his co-workers crystallized pepsin and trypsin and established them as proteins [ 3 ]. In subsequent years enzymology developed rapidly (table 1.1 ). The important developments during this period are: the elucidation of major metabolic pathways, such as the glycolysis and tricarboxylic acid cycle; the detection of numerous biochemical events of digestion, coagulation, muscular contraction and endocrine function, and their roles in the maintenance, control and integration of complex metabolic processes; the kinetic backgrounds to explain the observations of enzyme action and inhibition; and the development of protocols for examining the structures of functionally sensitive proteins. There has been exhaustive research on enzyme-catalyzed reactions and enzymes involved in cell metabolism. At present, 2000 different enzymes have been recognized, each of which catalyzes a different chemical reaction. Currently, more focus is being directed towards the application of enzymes. The high efficiency of enzymes makes them commercially valuable and their specificity of action is offering diverse advantages in clinical medicine.

Table 1.1.   Chronology of enzyme studies.

1.2. Properties of enzymes

Enzymes are the complex protein molecules, often called biocatalysts, which are produced by living cells. They are highly specific both in the reactions that they catalyze and in their choice of reactants, which are known as substrates. An enzyme typically catalyzes a single chemical reaction or a set of closely related reactions [ 4 ]. Side reactions resulting in the wasteful formation of by-products are rare in enzyme-catalyzed reactions, in comparison to uncatalyzed ones. Enzymes can also be defined as soluble, colloidal and organic catalysts that are produced by living cells, but are capable of acting independently of the cells [ 4 ]. Enzymes are currently being used in diverse areas in the food, feed, paper, leather, agriculture and textiles industries, resulting in major cost reductions. Simultaneously, rapid scientific progress is now encouraging the chemistry and pharmacological industries to embrace enzyme technology, a trend supported by concerns regarding energy, raw materials, health and the environment. One of the most common advantages of enzymes is their ability to function continuously even after their removal or separation from the cells. This means that even after the separation of cells from in vivo environments, they continue to work efficiently under in vitro conditions; we can conclude that these biocatalysts remain in an active state even after their isolation. Principally, enzymes are non-toxic, biodegradable and can be produced in ample amounts by micro-organisms for industrial applications. In this chapter, the isolation, production, purification, utilization and application of enzymes (in soluble and immobilized or insoluble form) are discussed in detail. Procedures such as recombinant DNA technology and protein engineering are frequently used to produce more efficient and beneficial enzymes. The industrial production and utilization of enzymes is an important part of industry. Interdisciplinary collaboration between areas such as chemistry, process engineering, microbiology and biochemistry is required to develop the best possible enzyme technology, and eventually to achieve increased production and maintain the enzyme's physico-chemical properties under in vitro environments.

For catalytic action, small quantities of an enzyme are sufficient, where this quantity of enzyme is much smaller in comparison to its substrates. The overall concentration of substrate transformed per mass of enzyme is often very large. Without exception, all enzymes are proteinaceous and exhibit all the properties of a protein. The treatment of enzymes by extreme temperature or extreme pH, or by treatment with other denaturing agents, results in the complete loss of catalytic activity. Structural configurations such as the primary, secondary, tertiary and quaternary structures of enzyme proteins are essential for their catalytic activity. The degree of catalytic activity chiefly depends on the integrity of the enzyme's structure as a protein. As per reports, enzymes have molecular weights ranging from about 12 000 to over 1 million Da. A number of enzymes consist only of polypeptides and contain no chemical groups other than amino acid residues, e.g. pancreatic ribonuclease. Numerous enzymes require a specific, heat stable, low molecular weight organic molecule, known as a co-enzyme. Moreover, a number of enzymes require both a co-enzyme and one or more metal ions for activity. A complete biochemically active compound is formed by the combination of a catalytically active enzyme (also called the protein part) with a co-enzyme or a metal ion—this is called a holoenzyme. The protein part of a holoenzyme is called an apoenzyme. In this arrangement a co-enzyme may bind covalently or noncovalently to the apoenzyme. In certain enzymes the co-enzyme or metal ion is only loosely and transiently bound to the protein. However, in others it is tightly and permanently bound, in which case it is known as a prosthetic group. A prosthetic group signifies a covalently bound co-enzyme. According to reports, co-enzymes and metal ions are stable under heating, while the protein part of an enzyme (the apoenzyme), is denatured by heat.

Prosthetic groups may be classified functionally into two major classes: co-enzymes and co-factors. Co-enzymes may be considered to be biosynthetically related to the vitamins, such as the co-enzyme nicotinamide adenine dinucleotide (NAD) which is vital for cellular energy metabolism and integrates the vitamin niacin into its chemical makeup. Moreover, a co-enzyme may be considered as a co-substrate, experiencing a chemical transformation throughout the enzyme reaction (NAD is reduced to NADH), the reversal of which requires a separate enzyme, perhaps from a different cellular site. Co-enzymes might thus travel intra-cellularly between apo-enzymes and, by transferring chemical groupings, integrate several metabolic processes. Table 1.2 shows a list of the more common co-enzymes and their functions. In contrast to co-enzymes, co-factors, such as pyridoxal phosphate or hem groups, remain with one enzyme molecule and in conjunction complete a cycle of a chemical change brought about by one enzyme turnover [ 5 ]. Other enzymes, such as carboxypeptidase, require metal ions as co-factors, the divalent cations Mg 2+ , Zn 2+ and Mn 2+ being the most common; these are often called enzyme activators [ 6 ]. Table 1.3 lists several enzymes and their respective co-factors.

Table 1.2.   Several co-enzymes employed in the transfer of specific atoms or functional groups.

Table 1.3.   Several enzymes and their co-factors.

1.3. Catalysis

The role of a catalyst is to increase the speed of a chemical reaction. When the rate of a chemical reaction is governed by a soluble catalyst, which may result in a further increase in the rate of chemical reaction, it is called homogeneous catalysis. In this case catalysis occurs in a solution. When the catalyst is in a separate phase from the reactants, or when catalysis occurs on a insoluble surface or an immobilized matrix, it is known as heterogeneous catalysis. Enzymes are also called biological catalysts. These biological catalysts generally have the properties of homogeneous catalysts, however, a number of enzymes present in membranes are insoluble, and thus are called heterogeneous catalysts. Enzyme specificity is the absolute specificity of protein catalysts to identify and bind to only one or a few molecules. In this process the enzyme carries a defined arrangement of atoms in their active site to bind with the substrate. This active site on the enzyme should have a shape that accurately matches the substrates. Thus specificity is achieved when an enzyme with an active site binds with the chemical reactants (the substrates) at their active sites via weak bond interactions. To undergo a chemical reaction, this active site carries certain residues that form a temporary bond with the chemical reactants, termed the binding site, whereas the catalytic site carries the residues that are responsible for catalysis. Specificity is achieved when a substrate binds to an enzyme that has a defined arrangement of atoms in the active site. An enzyme always catalyzes a single type of chemical reaction, which involves the formation and breakdown of covalent bonds. Since they are specific to one particular reaction, this feature of enzymes is called reaction specificity, also known as absolute reaction specificity, i.e. no by-products are formed.

1.4. The structure of enzymes

Enzymes always act as catalysts and small quantities compared to their substrate are required to considerably increase the rate of chemical reactions, wherein the enzymes themselves experience no overall change [ 7 , 8 ]. In contrast to all true catalysts, an enzyme does not alter the ultimate equilibrium position of a reaction, which is thermodynamically determined, thus merely the rate of completion of equilibrium of a feasible reaction is augmented. In addition to catalytic properties, enzymes exhibit the physico-chemical behavior of proteins: their solubility, electrophoretic properties, electrolytic behaviors and chemical reactivity [ 7 , 8 ]. The primary structural configuration and catalytic action of enzymes is determined by the linear chain of amino acid residues linked via peptide bonds, which constitute a protein molecule. Localized folding of the primary structure is called a secondary structure, whereas the complete folding of the molecule is known as a tertiary structure. In contrast to these structural configurations, a quaternary structure is the agglomeration of several folded chains. The structural features of enzymes are shown in figures 1.1 and 1.2 . In contrast to traditional chemical catalysts, e.g. hydrogen ions, heavy metals or metal oxides, which are most effective in organic solvents, at very high temperatures or at extreme pH values, enzymes operate most efficiently under very mild conditions. When using enzymes, there are certain issues that require attention, such as deviation from homogeneous aqueous solutions, physiological pH and temperature, which can rapidly destroy enzyme activity. However, under normal conditions the increase in reaction rate is rarely matched by their non-protein counterparts.

Figure 1.1.  Structural features of enzyme.

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Figure 1.2.  Principle components of an enzyme.

1.5. Structural features: primary and secondary structures

Three-dimensional analysis of the amino acid sequence of lysozyme of hen's egg white has demonstrated some features essential for primary structure [ 9 , 10 ]. These are:

• •   Molecules derived from a similar source have a similar order of amino acid residues and appear to be random with no obvious predictability.
• •   Even though numerous enzymes are intramolecularly crosslinked via disulfide bridges of cysteine, no branching occurs.

Current databases suggest that a small number of amino acids are extra and most are 'functional', i.e. the majority of them co-operatively control the higher orders of structural organization and therefore the catalytic activity. When comparing the primary structures of enzymes performing similar functions, wide structural homologies are detected in their sequence, mainly in the patterns of their nonpolar residues. For example, pancreatic juice contains five inactive precursors (zymogens), namely chymotrypsinogen A, B and C, trypsinogen and proelastase; all of these are activated to the respective proteases by proteolytic cleavage [ 11 ].

1.6. The mechanism of action of enzymes

The mechanism of action is based on a chemical reaction, in which the enzyme binds to the substrate and finally forms an enzyme–substrate complex. This reaction take place in a relatively small area of the enzyme called the active or catalytic site. In other words, the mechanism of enzyme action is based on the nature of the enzyme–substrate interaction, which accounts for the reaction specificity of the biological catalysts. The active or catalytic site of an enzyme is constituted by several amino acids, located at some distance from each other in the peptide chain. These amino acids are brought close together by the folding resulting from the secondary and tertiary structure of the enzymes. Side chains of amino acid residues at the catalytic site provide groups for binding with specific groups of the substrate. Co-factors assist the catalysis. The substrate forms bonds with amino acid residues in the substrate binding domain of the active site. The binding induces a conformational reaction in the active site. During the reaction, the enzyme forms a transition-state complex. As the products of the reaction disassociate, the enzyme returns to the original state. Two different models postulated for the mechanism of enzyme action are given below.

1.6.1. The Fisher template model (lock and key model)

This is a rigid model of the catalytic site, proposed by Emil Fischer in 1894 [ 12 ]. The model explains the interaction between a substrate and an enzyme in terms of a lock and key analogy. In this model, the catalytic site is presumed to be preshaped. The substrate fits as a key fits into a lock. The drawback of this model is the implied rigidity of the catalytic site. The model cannot explain changes in enzyme structure in the presence of allosteric modulators.

1.6.2. Induced fit model

In contrast to the above method, this model suggests a flexible mode for the catalytic site. To overcome the problems of the lock and key model owing to the rigid catalytic site, Koshland [ 13 – 15 ] suggested an induced fit model in 1963. The important feature of this procedure is the flexibility of the active site. In the induced fit model, the substrate induces a conformational change in the active site of the enzyme so that the substrate fits into the active site in the most convenient way so as to promote the chemical reaction. This method suggests competitive inhibition, allosteric modulation and inactivation of enzymes on denaturation.

The Michaelis–Menten theory of enzyme action [ 16 ] offers the basis for most current research on the mechanism of enzyme action. This concept of the enzyme–substrate complex scheme assumes the combination of the enzyme and substrate in phase one (occasionally known as the transition phase) of the enzyme activity and liberation of the enzyme and the products of the catalysis in phase two of the reaction.

1.6.3. Covalent catalysis

Covalent catalysis is evidenced in enzymes capable of forming covalent bonds between the substance and the catalytic group of the active site [ 17 ]. A number of enzymes react with their substrates to form very unstable, covalently joined enzyme–substrate complexes, which undergo further reaction to yield products much more readily than in an uncatalyzed reaction. Several of the enzymes that exhibit covalent catalytic behavior are listed in table 1.4 .

Table 1.4.   Various enzymes exhibiting covalent catalytic behavior.

1.7. Catalysis via chymotrypsin

Hummel and Kalnitzky suggested an enzyme mechanism through the depiction of the sequential transition states experienced by the enzyme–substrate complex during catalysis [ 18 ]. Chymotrypsin is a digestive enzyme, responsible for proteolysis (breakdown of proteins and polypeptides) in the duodenum. Chymotrypsin favorably breaks peptide amide bonds (the carboxyl side of the amide bond is a large hydrophobic amino acid). These amino acids contain an aromatic ring in their side chain that fits into a 'hydrophobic pocket' of the enzyme. It is stimulated in the presence of trypsin. Trypsin and chymotrypsin are both serine proteases with high sequence and structural similarities, but with different substrate specificity [ 19 , 20 ].

1.7.1. Intermediary stages of chymotrypsin

As discussed above, chymotrypsin is a protease enzyme that cuts on the C-terminal phenylalanine, tryptophan and tyrosine on peptide chains [ 21 ]. Additionally, it is more specific for aromatic amino acids because of its hydrophobic pocket. Comparable to other serine proteases, chymotrypsin also catalyzes the hydrolysis of certain esters [ 22 ]. The molecular events involved in catalysis are called intermediary enzymology. Chymotrypsin, a protease, favorably accelerates breakdown of peptide bonds in which the aromatic amino acid (Phy, Try, or Trp) or bulky nonpolar R group (Met) contribute a carboxyl group. The synthetic substrate p-nitrophenyl acetate allows colorimetric analysis of chymotrypsin activity, as hydrolysis to p-nitrophenol, which is alkali, changes into the chromophore anionic forms.

1.7.2. Kinetic behavior of α -chymotrypsin

The kinetics of chymotrypsin of p-nitrophenyl acetate can be considered in a 'stop-flow' apparatus. This procedure utilizes substrate quantities of enzymes and measures the events in the first few milliseconds [ 23 ]. The use of p-nitrophenyl acetate as a substrate offers the prospect of investigating solvent effects on both the acylation of the enzyme and the hydrolysis (deacylation) of the acyl enzyme [ 23 ]. The significant features of the slow-flow kinetics of chymotrypsin are:

• - a burst phase featuring rapid liberation of an anion.
• - a subsequent 'steady-state' phase, with slower release of extra anion.
• •   A 'charge relay network' acts as a proton shuttle during catalysis by chymotrypsin. The charge relay network of chymotrypsin encompasses three aminoacyl residues that are far apart in a primary structural sense, but close together in a tertiary structural sense. While most of the charged residues of chymotrypsin are present at the surface of the molecule, those of the charge relay network are hidden in the otherwise nonpolar inner side of the protein. These charges transmit residues which activate sequential proton shifts that shuttle protons in the opposite direction. An equivalent series of proton shifts is assumed to accompany the hydrolysis of the physiologic chymotrypsin substrate, e.g. a peptide.

1.7.3. Selective proteolysis in creation of the catalytic sites of enzymes

Various enzymes, hormones and other physiologically active proteins are produced as inactive precursors (zymogens) that are further transformed to the active form by selective enzymatic cleavage (limited proteolysis) of peptide bonds. The final step to activating enzymatic function is limited proteolysis, either in a single activation step or in a consecutive series (cascade). The specificity of each activation reaction is evaluated by the complementarity of the zymogen substrate and the active site of the attacking protease. The arrangement of successive activation reactions is controlled by the specificity of each enzyme, while the extent of amplification of the initial stimulus is evaluated by the effectiveness of each activating step. Zymogen activation produces a prompt and irreversible response to a physiological stimulus, and is capable of initiating new physiological functions. Classical examples are the processes of hormone production, fibrinolysis, complement activation, blood coagulation, supra-molecular assembly, metamorphosis, fertilization and digestion. The zymogens of the pancreatic serine proteases, in particular, have functioned as models for detailed studies of the nature of the molecular changes that are involved in the intense increase in enzymatic activity that results upon incomplete proteolysis of the zymogen.

Specific proteolysis is a common means of activating enzymes and other proteins in biological systems. A number of proteins are manufactured and released in the form of inactive precursor proteins called proproteins. Various enzymes attain full enzymatic activity as they suddenly fold into their characteristic three-dimensional forms. In contrast, other enzymes are produced as inactive precursors that are successively activated by breakdown of one or a few specific peptide bonds. The inactive precursor is known as a zymogen (or a pro-enzyme). In other words, when the proteins are enzymes, the proteins are called pro-ezymes or zymogens (table 1.5 ). An energy source (ATP) is not required for cleavage [ 11 ]. Thus, in comparison to reversible regulation by phosphorylation, even proteins sited outside cells can be triggered by this means. An additional noteworthy difference is that proteolytic activation, in comparison with allosteric control and reversible covalent modification, occurs just once in the life of an enzyme molecule. Transformation of a proprotein to the mature protein includes selective proteolysis. This transforms the proproteins by one or more consecutive proteolytic clips to a arrangement in which the individual activity of the mature protein (its enzymatic activity) is expressed, e.g. the hormone insulin (proinsulin), the digestive enzyme chymotrypsin (chymotrypsinogen), a number of factors for blood clotting and for the blood clot dissolution cascades, and the connective tissue protein collagen (procollagen). Chymotrypsinogen consists of 245 amino acid residues, and is practically devoid of enzymatic activity. As the reaction starts, it is converted into a fully active enzyme. This occurs when the peptide bond joining arginine 15 and isoleucine 16 is cleaved by trypsin. The subsequent active enzyme, known as π-chymotrypsin, then acts on other π -chymotrypsin molecules. Two dipeptides are eliminated to form α -chymotrypsin (the stable form of the enzyme) [ 11 ]. The three subsequent chains in α -chymotrypsin remain interconnected to each another by two interchain disulfide bonds. The outstanding feature of this process is that cleavage of a single specific peptide bond alters the protein from a catalytically inactive form into one that is fully active. The transformation of prochymotrypsin (Pro-CT), a 2,4,5-aminoacyl residue polypeptide, to the active enzyme α -chymotrypsin includes three proteolytic clips and the formation of an active intermediate called π -chymotrypsin ( π -CT) and consequently to the mature catalytically active enzyme α -chymotrypsin ( α -CT). Examples of gastric and pancreatic zymogens are listed in table 1.5 .

Table 1.5.   Gastric and pancreatic zymogens.

1.7.4. Kinetic models for enzymes

Generally, enzyme kinetics is defined as the study of the rate of reactions, i.e., how the substrate concentration impacts the velocity of the reaction. Enzyme kinetics involves optimization of bio-catalytic reactions to allow process design and scaling up processes to further increase the production and minimize the overall overhead costs of various procedures. Kinetic investigations in the branch of biochemistry concerned with enzymes can be categorized into three types:

• •   Transient-state kinetics : This is the stage of reaction before the steady or rapid-equilibrium state, and involves quick reactions between the enzymes and substrate. These sudden changes in the reaction mixture when the substrate and enzymes are mixed require advance equipment to monitor the reaction before it changes into the steady state. The mechanisms of the reaction are associated with the enzyme structural configuration. Basic steps are involved during an enzyme-catalyzed reaction, which allow the direct study of the intermediates and products formed during a single enzyme cycle, which may further help in direct analysis of individual reaction steps for short times. In this type of reaction a sufficient concentration of enzymes is used to witness the intermediate and product formation.
• •   Steady-state kinetics : This is the phase in which the rate of formation of intermediates and the rate of decomposition remain the same, and thus the concentrations of reactive intermediates remain the same. During this reaction substrate concentration is greater than enzyme concentration. The Michaelis–Menten enzyme kinetic (figure 1.3 ) can be considered as the most often studied reaction for several enzymes. For example, chymotrypsin (protease) with a high concentration of substrate achieves maximum velocity of the reaction (called the first order of reaction) but at a certain point the substrate occupies all binding sites of the enzyme, after which further addition of substrate does not increase the rate. This is called the zeroth order of reaction (the steady state). It is the phase in which the enzyme and substrate concentrations cannot be determined using the dissociation constant. Thus steady-state enzyme kinetics is based on the theory that a catalytic reaction remains constant if the reaction is not exposed to continuous changes.
• •   Rapid-equilibrium kinetics : This the phase in which both the enzyme and substrate concentrations can be determined using the dissociation constant. During this procedure total enzyme concentration remains constant during the reaction and the concentration is very small compared to the amount of substrate. In this reaction, before the rate-determining reaction, the reactions are in equilibrium with their components, thus this stage is called rapid-equilibrium kinetics.

Figure 1.3.  The Michaelis–Menten enzyme kinetic.

According to reports, factors that affect enzyme-catalyzed reactions also affect the velocity of a reaction. These factors are called modifiers of enzyme-catalyzed reactions. These modifiers can be divided into two classes: inorganic modifiers (enzyme activators) and organic modifiers (enzyme inhibitors). These factors can have different types of effects on the velocity of the reaction; nevertheless the most vital effect is that they offer many pathways to products, e.g. when one modifier is bound to an enzyme, it alters the rate of reaction and thus forms two rate constants. However, when two modifiers participate, there are five self-regulating equilibria, resulting in three paths for making products.

There are two mechanisms, single-substrate and multiple-substrate, that are helpful in studying the different stages of enzymatic reactions. Understanding these stages helps in understanding the properties of enzymes. Certain enzymes have single substrates (a single substrate binding site), e.g. triosephosphate isomerase, whereas certain enzymes have multiple substrates molecules (multiple binding sites), such as dihydrofolate reductase, and bind with multiple substrates. After the exploration of specific RNA sequences required for RNA replication, new biocatalysts in the form of ribozymes have emerged with the potential to catalyze specific biochemical reactions. There is a misconception about biological catalysts that all biological catalysts are made up of proteins, which is not true; some are RNA-based catalysts (ribozymes and ribosomes). Both are important for many cellular functions. A major difference between enzymes and ribozymes is that RNA-based catalysts are restricted to only a few reactions; however, their reaction mechanisms and kinetics can be studied and classified by similar procedures. Enzyme-based mutation, in particular site-directed mutagenesis, is an important approach to alter genes and investigate the functional and structural features of enzymes, e.g. mutation of the enzyme present in Coprinus cinereus peroxidase offers an understanding of its increased thermostability. Challenges involved in studying cascades of reactions catalyzed by a multi-enzyme, e.g. proteasome involved in the ubiquitin–proteasome pathway, can be overcome by establishing understanding of the complex structure and the respective biochemical reactions. This understanding allows exploration of active sites, intermediate compounds, final products and their interrelation with complex machinery, as well as biochemical reactions. It has been well understood that enzymes that accelerate complex reactions have numerous substrates and involve complex enzyme kinetic mechanisms. As discussed above, most of the biochemical reactions occurring in the body are multi-substrate reactions. In such reactions two substrates are involved and yield two products (figure 1.4 ). These types of reactions involve the transfer of a compound from one compoment to another, e.g. when glucose reacts with ATP in the presence of hexokinase it forms glucose 6-phophaste and ADP. Here, phosphate from ATP is transfered to glucose to form glucose 6 phosphate. The mechanism of catalysis involves two types of reactions: sequential and non-sequential reactions. Sequential reaction results in the formation of a ternary complex. This means that both of the substrates involved in the reaction bind with an enzyme to form the product (figure 1.4 ). Sequential reaction is further divided into two types: the random and compulsory order mechanisms. As the name suggests, in a 'random' mechanism, either substrate can bind first and any product can leave first. In contrast to the random order mechanism, in the compulsory order mechanism the order of binding of the substrate and order of release of the product is specific; this is also called the Theorell–Chance mechanism (figure 1.4 ). In a non-sequential reaction, also called the 'ping-pong' mechanism, formation of ternary complex does not take place. In these types of reactions, when the first substrate binds with enzyme its product is released, and then the second substrate binds and its product is released. Such a reaction is called a double placement reaction. Thus only a single substrate binds at a time; this may be due to the presence of a single binding site on the enzyme. Major differences between the sequential and non-sequential reactions are that the formation of a ternary complex takes place only in the sequential reaction, and that in the sequential reaction both substrates bind to the enzyme and release products, while in the non-sequential mechanism the substrates bind and release their products one after the other (figure 1.4 ).

Figure 1.4.  Multi-substrate reactions.

Another type of sequential mechanism is the systematic mechanism, which involves the addition of substrates and formation of products in a specific order.

1.7.5. Enzyme mediated acid–base (general) catalysis

Several protein enzymes use general acid–base catalysis as a way to increase reaction rates [ 26 ]. The amino acid histidine is optimized for this function because it has a pK(a) (where K(a) is the acid dissociation constant) near physiological pH [ 26 ].

When the substrate has been bound at the catalytic site, the charged functional groups of the side chains of neighboring aminoacyl residues may contribute in catalysis by behaving as acidic or basic catalysts. There are two extensive groups of acid–base catalysis by enzymes: general and specific (acid or base) catalysis. Specific acid or specific base catalysis are those reactions in which the reaction rates fluctuate under the influence of changes in H + or H 3 O + concentration, but are independent of the concentrations of the other acids or bases present in the solution. In contrast to specific catalysis, general acid or general base catalysis are the reactions whose rates are very reactive to all acids (proton donors) or bases (proton acceptors) present in the solution. To examine whether a given enzyme-catalyzed reaction is a general or specific acid or base catalysis, the rate of reaction is determined under two sets of circumstances:

• •   at different pH values at a constant buffer concentration, and
• •   at constant pH values but at different buffer concentrations. Against this background, if the degree of the reaction deviates as a function of pH at a constant buffer concentration, the reaction is specific base/acid catalyzed if the pH is above/below 7.0. If the reaction rate at a constant pH rises as the buffer concentration increases, the reaction is general base/acid catalysis, if the pH is above/below 7.0.

1.7.6. Metallozymes

Almost 25% of all enzymes include tightly bound metal ions or need them for activity. The major role of these metal ions is investigated using techniques such as x-ray crystallography, magnetic resonance imaging (MRI) and electron spin resonance (ESR). A metalloprotein is a protein that contains a metal ion co-factor. Metallozymes contain a certain amount of functional metal ion that is retained during the course of purification [ 27 ]. A metal-activated enzyme binds with metals less firmly, but needs to be activated by addition of metals. Four types of complexes are possible for the tertiary complexes of the catalytic site (Enz), a metal ion (M) and substrate (S) that exhibit 1:1:1 stoichiometry:

All of these complexes are possible for metal-activated enzymes. Metallozymes cannot form the EnzSM complex (substrate–bridge complexes), as the purified enzyme exists as Enz–M. Three generalization can be made:

• •   The majority of the kinases (ATP: phosphotransferases) form substrate–bridge complexes of the type enzyme–nucleotide–M.
• •   Phosphotransferases (phosphoenolpyruvate or pyruvate used as the substrate), enzymes catalyzing other reactions of phosphoenolpyruvate and carboxylases, form metal bridge complexes (Enz–M–S).
• •   A particular enzyme may form one type of bridge complex with one substrate and a different type with another.

The metal ions participate in each of the four mechanisms by which the enzymes are known to accelerate the rates of chemical reaction:

• •   Approximation of reactants.
• •   Covalent catalysis.
• •   General acid–base catalysis.
• •   Induction of strain in the enzyme or substrate.

Metal ions are electrophiles (attracted to electrons) and share an electron pair forming a sigma bond. They may also be considered as super acids as they exist in neutral solutions, frequently having a positive charge which is greater than their quantity. Mn 2+ , Ca 2+ and Mg 2+ are the metal ions that are most commonly used in enzymatic catalysis. Two metal ions, iron and manganese are used in the form of haemprotein. Metal ions have the potential to accept electrons via sigma or pi bonds to successively activate electrophiles or nucleophiles. By means of donating electrons, metals can activate nucleophiles or act as nucleophiles themselves. The co-ordination sphere of a metal may bring together the enzyme and substrate or form chelate-producing distortion in either the enzyme or substrate [ 28 ]. A metal ion may also mask a nucleophile and thus avoid an otherwise probable side reaction. Metals can also function as three-dimensional templates for the co-ordination of basic groups on the enzyme or substrate.

1.8. Enzyme inhibition

Enzyme inhibition decreases the activity of an enzyme without significantly disrupting its three-dimensional macromolecular structure. Inhibition is therefore distinct from denaturation and is the result of a specific action by a reagent directed or transmitted to the active site region. When low molecular weight compounds interfere with the activity of enzymes by partially reducing or completely inhibiting the enzyme activity either reversibly or irreversibly, it is known as enzyme inhibition. The compounds responsible for such inhibition are called enzyme inhibitors. To protect the enzyme catalytic site from any change, a ligand binds with a critical side chain in the enzyme. Chemical modification can be performed to test the inhibitor for any drug value. Studies of enzymes can yield much information about the following:

• •   A number of drugs useful in medicine, which seem to function because they can inhibit certain enzymes in malfunctioning cells.
• •   The convenience of elucidating metabolic pathways in cells.
• •   The mechanism of the catalytic activity.
• •   The nature of the functional group at the active site.
• •   The substrate specificity of the enzyme.

The pharmacological action of drugs is mainly based on enzyme inhibition, e.g. sulfonamides and other antibiotics. In the majority of cases the enzyme inhibited is known. The development of nerve gases, insecticides and herbicides is based on enzyme inhibition studies. There are two major types of enzyme inhibition: reversible and irreversible.

Reversible inhibitors efficiently bind to enzymes by forming weak non-covalent interactions, e.g. ionic bonds, hydrophobic interactions and hydrogen bonds. Reversible inhibitors do not form any strong chemical bonds or reactions with the enzyme, they are formed quickly and can easily be removed, in contrast to irreversible inhibitors. Reversible inhibition includes competitive inhibition, uncompetitive inhibition and noncompetitive inhibition. Irreversible inhibition includes group specific inhibition (reacts only to a certain chemical group), reactive substrate analogs (affinity label) and inhibitors that are structurally similar to the substrate and will bind to the active site, and mechanism-based inhibitors (enzymes transform the inhibitor into a reactive form within the active site).

1.9. Pharmaceutical applications

Currently, enzymes are often utilized for a broad range of applications such as: washing powders (e.g. proteases, lipases, amylases); textile manufacture (amylases and catalase to remove the starch); the leather industry (proteases to hydrolyze proteins); the paper industry; improvement of the environment; food production (enzyme-modified cheese/butter), processing (glucose oxidase for dough strengthening) and preservation; and medical applications. According to current reports, several enzymes are produced industrially and there are significant applications in the food industry (45% of use), detergent industry (35%), textiles industry (10%) and leather industry (3%). Details on the applications of individual enzymes are provided in table 1.6 .

Table 1.6.   Industrially produced enzymes from plant sources and their applications.

1.9.1. Diagnostic applications of enzymes

Enzymes have been used widely in diagnostic applications varying from immunoassays to biosensors. Enzyme immunoassay methods hold great promise for application under a wide variety of conditions. Under laboratory conditions they can be as sensitive as radio-immunoassays, but they can also be adapted as simple field screening procedures [ 29 , 30 ]. The examination of enzyme quantity in the extracellular body fluids (blood plasma and serum, urine, digestive juices, amniotic fluid and cerebrospinal fluid) are vital aids to the clinical diagnosis and management of disease. Most enzyme-catalyzed reactions occur within living cells, however, when an energy imbalance occurs in the cells because of exposure to infective agents, bacterial toxins, etc, enzymes 'leak' through the membranes into the circulatory system. This causes their fluid level to be raised above the normal cell level. Estimation of the type, extent and duration of these raised enzyme activities can then furnish information on the identity of the damaged cell and indicate the extent of injury. Enzyme assays can make an important contribution to the diagnosis of diseases, as a minute change in enzyme concentration can easily be measured. Determination of the changes in enzyme level thus offers a greater degree of organ and disease differentiation in comparison to other possible clinico-chemical parameters, e.g. albumin or gamma globulin. Currently, the diagnostic specificity of enzyme tests is such that they are limited primarily to confirming diagnosis, offering data to be weighed alonside other clinical reports, owing to lack of disease specific enzymes. Table 1.7 includes a number of diagnostically important enzymes which are most often examined in clinic laboratories [ 29 , 30 ].

Table 1.7.   Diagnostically significant enzymes.

1 B, brain; E, erythrocytes; H, heart muscle; Ht, hepatobiliary tract; I, intestinal mucosa; K, kidney; L, M, skeletal muscle; Pa, pancreas; P1, placenta; Pr, prostate gland; S, saliva.

1.9.1.1. Enzyme examinations in diseases of the liver and biliary

The diseases of the liver and gastrointestinal tract were among the first to which serum enzyme tests were applied. They have proved to be most effective owing to the large size of the organs and the wide range and abundance of enzymes [ 32 – 36 ]. The liver-based enzymes GOT, GPT and AP are examined to evaluate the site and nature of liver disease. LD, GGT, OCT and CHE are also examined. Several enzymes employed in the diagnosis of liver diseases along with their respective levels are listed in table 1.8 .

Table 1.8.   Liver diseases and enzymes used in diagnosis [ 32 – 36 ].

1.9.1.2. Enzyme applications in heart disease

According to previous reports, no single enzyme has yet been reported to cure myocardial damage. The discovery of serum glutamine oxalacetic acid transaminase determination (GOT) in 1954 was considered a significant step forward in the diagnosis of acute myocardial infarction. A mixture of results from assays of CPK (creatine phosphokinase), HBD ( α -hydroxybutyrate dehydrogenase) and GOT (glutamine oxalacetic acid transaminase)—each of which has been shown to be elevated in more than 90% of cases—is used for diagnostic purposes [ 37 – 39 ]. The level of CPK starts rising three to four hours after the initial onset of pain, followed in order by GOT and AST (HBD) which appear after approximately eight hours. The maximum levels are reached in the same sequence, CPK after 24 h, LD 1 after 36 h and AST after about two days. The rise in enzyme levels is fairly moderate, AST and CPK increase by four to ten times their respective normal levels and LD 1 is approximately five-fold higher than normal. An enzyme known as hyaluronidase (hyaluronate hydrolysis) has been reported to cure heart attack [ 38 ]. The activity of many enzymes including aldolase, malic dehydrogenase, isomerase and ICD may increase following myocardial infarction [ 38 ].

1.9.1.3. Diagnosis of muscle disease

Skeletal muscle disorders include diseases of the muscle fibers (myopathies) or of the muscle nerves (neurogenic disorders) [ 40 ]. In myopathies CPJ, LD, ALD, GOT and GPT levels are raised. In the case of neurogenic diseases and hereditary diseases, CPK is occasionally raised (2–3 fold) [ 40 ]. Damage to the muscle may be due to extensive muscular exercise, drugs, physical trauma, inflammatory diseases, microbial infection or metabolic dysfunction, or it may be genetically predisposed. In muscular disorders the level of CPK is elevated in serum with the highest frequency and is assayed in the diagnosis of these disorders. An additional useful assayed enzyme is acetyl cholinesterase (AChE), which is significant in regulating certain nerve impulses [ 41 ]. Various pesticides affect this enzyme, so farm labors are frequently tested to be sure that they have not received accidental exposure to significant agricultural toxins. There are number of enzymes that are characteristically used in the clinical laboratory to diagnose diseases. There are highly specific markers for enzymes active in the pancreas, red blood cells, liver, heart, brain, prostate gland and many of the endocrine glands [ 41 ]. From the time when these enzymes became comparatively easy to examine using automated techniques, they have been part of the standard blood tests that veterinarians and medical doctors are likely to need in the diagnosis and treatment/management of diseases.

1.9.2. Enzymes in therapeutics

Enzymes have two significant features that differentiate them from all other types of drugs. First, enzymes frequently bind and act on their targeted sites with high affinity and specificity. Second, enzymes are catalytic and convert numerous target molecules to the desired products. These two important features make enzymes specific and potent drugs that can achieve therapeutic biochemistry in the body that small molecules cannot. These features have resulted in the development of many enzyme-based drugs for a wide range of disorders [ 42 ]. Currently, numerous enzymes are used as therapeutic agents, owing to the following features:

• •   High specificity to their substrates.
• •   Proficient in producing the desired effect without provoking any side effects.
• •   Water soluble.
• •   Extremely effective in a biological environment.

Enzymes as therapeutic agents also have some serious disadvantages which restrict their application. Their bulky structure, due to their large molecular weight, excludes them from the intracellular domain. Owing to their high proteinaceous nature they are highly antigenic and are rapidly cleared from blood plasma. Extensive purification from pyrogens and toxins is essential for parenteral enzymes, which increases the cost. Table 1.9 lists some therapeutically important enzymes.

Table 1.9.   Therapeutically important enzymes.

1.9.2.1. Enzyme therapy of cancer

In traditional medicine, proteolytic enzymes derived from plant extracts have been used for a long time In addition to proteolytic enzymes from natural resources such as plants, 'modern' enzyme therapy includes pancreatic enzymes. Therapeutically, the use of proteolytic enzymes is partly based on scientific reports and is partly empirical [ 43 ]. Clinical evidence of the use of proteolytic enzymes in cancer studies has typically been obtained with an enzyme preparation comprising a combination of papain, trypsin and chymotrypsin. Earlier reports proved that enzyme therapy can reduce the adverse effects caused by radiotherapy and chemotherapy. There is also a report available that, in some types of tumors, survival may be sustained. The positive effects of systemic enzyme therapy appear to be based on its anti-inflammatory potential. Nevertheless, the exact mechanism of action of systemic enzyme therapy remains unsolved. The proportion of proteinases to antiproteinases, which is regularly used as a prognostic marker in cancer studies, is likely to be influenced by the oral administration of proteolytic enzymes, most likely via induction of the synthesis of antiproteinases. In addition, there are many alterations of cytokine composition during treatment with orally administered enzymes, which might be a sign of the efficacy of enzyme therapy [ 44 ].

Proteases and their inhibitors have long been studied in several tumor systems. However, out of numerous promising serine and metalloproteinase inhibitors, not a single one is included in oncology at present. The present exploration for active antiproteolytic agents is in contrast to the traditional approach, as evidenced by John Beard, who proposed the management of advanced cancer using fresh pancreatic extracts whose antitumor activity was based on their proteolytic potential.

The enzymatic treatment of tumors is based on the idea of denying the abnormal cells their essential metabolic precursors such as amino acids, nucleic acids and folates. A number of enzymes have been examined and evidenced as antitumor agents. l -serine dehydratase, l -arginase, carboxypeptidase G (folate depletion), l -asparaginase, l -methioninase, l -phenylalanine ammonia lyase, l -glutaminase, l -tyrosinase and xanthine oxidase have been studied for their anticancer activity. Enzyme preparations such as asparaginase (amidase), bromelain (protease) and chymotrypsin (protease) have also been studied as cancer treatments (table 1.9 ).

l -asparaginase is the most widely investigated enzyme. It has been reported in treatment against three neoplastic diseases, acute lymphoblastic leukemia, leukemic lymphosarcoma and myeloblastic leukemia. It deprives the cancerous cells of their nutritional asparagine supply. Asparagine is essential for protein synthesis, which takes place inside the cell, and decreased protein synthesis perhaps accounts for the immunosuppression and toxic effects of asparaginase-based treatment.

The prospects of enzyme-based treatment against cancer are very bright, but the difficulties of antigenicity and short circulation time remain to be overcome.

1.9.2.2. Enzymes in thrombolytic treatment

Activation of the blood clotting mechanism during inflammation is part of the body's defense mechanism which requires therapeutic intervention. Under normal physiological conditions there is an equilibrium between blood coagulation (clotting) and fibrinolysis (the process of dissolving the clotted blood) [ 47 ]. Biocatalysts such as enzymes, ribozymes, pro-enzymes, activators and pro-activators are responsible for maintaining equilibrium between clot formation and fibrinolysis. Imbalances in the concentration of these bio-activators may disturb physiology. In the biological process of fibrogenesis, clot formation takes place due to the plasma protein (soluble fibrinogen), which is ultimately converted to insoluble fibrin by the enzyme thrombin. This process is dependent on the conversion of thrombin from prothrombin. This bio-conversion takes place after the cascade of enzymatic reactions which involved certain key biological compounds called clotting factors. A blood clot dissolving enzyme known as plasmin is present in the blood as the pro-enzyme plasminogen. During clot dissolution activators convert the plasminogen to plasmin. This biological process is well regulated by certain process such as vasoconstriction, formation of a fibrin and clot platelet aggregation [ 46 ].

As the body utilizes enzymes in conserving this key balance of homeostasis, in a similar way we can utilize enzymes to repair or restore the homeostatic balance once it is lost. Several reports have shown that one of the best approaches for treating such clinical conditions is the administration of enzymes capable of converting plasminogen to plasmin (the enzyme which dissolves the clot) via intraveneous injection. This type of treatment is called therapeutic thrombolysis or thrombolytic therapy. In this treatment, pharmacological agents are used to medically induce clot breakdown [ 47 ]. Various novel thrombolytic agents have been derived from different sources for therapeutic use, such as from bacteria (streptokinase), the venom of the Malayan pit viper (Arvin), a filamentous fungus Koji mold Aspergillus oryzae (brinase), a South American snake (reptilase) and human urine (urokinase) [ 47 ].

Current advancements in thrombolytic therapy are more focused on the treatment of occlusions (blockages) of blood vessels. These types of therapy can be considered as life-saving and emergency medicine for life-threatening conditions such as myocardial infarction and massive pulmonary embolism, which are the most common reasons for cardiac arrest. This life-saving treatment is more reliable in preventing the blockages of vessels in the lungs and heart. Artery blockage conditions such as pulmonary embolism in the lungs by the formation of a clot creates tension on the right side of the heart, resulting in shortness of breath and chest pain mainly upon breathing in. Enzyme-based thrombolysis for treating massive pulmonary embolism has been considered as an effective approach to dissolving clots in these large vessels. Since surgical removal raises the chances of new blood clot formation that can cause another pulmonary embolism at the same or a different site, it is considered a dangerous practice and thrombolytic therapy is considered the more effective treatment [ 47 ]. Nevertheless, reoccurrence of clot formation or clot re-formation is very common in patients who have undergone enzyme-based thrombolytic treatment. Researchers from various organizations (1971) determined the effectiveness of streptokinase over heparin in reducing the chances of death in acute myocardial infarction patients. Significant results were obtained during this experiment. As discussed above, re-formation of the clot is one of the major concerns in fibrinolytic therapy. Most clinicians start treatment with a high dose of fibrinolytic agents, which is reduced later on. This approach may reduce disease progression for some time, but often increases the chances of clot re-formation. Even after the dissolution of the clot it is very difficult to maintain the same physiologically balanced environment (homeostasis) at the site of damaged tissues and the chance of new clot formation at that particular location is very high. Therefore, fibrinolytic based treatment is always accompanied by anticoagulants, such as heparin [ 46 ].

Major concerns associated with streptokinase therapy are fever, a tendency for bleeding, antigenicity (as with any foreign protein) and the difficulty of determining the proper dose [ 47 ]. Post-enzymatic treatment bleeding is one of the major concerns and it is also a concern when anticoagulants are used alone. According to current research, urokinase (produced in the kidneys and obtained from human urine) is considered safer than streptokinase. For the production of urokinase, 2300 l of urine is required to yield only 29 mg of purified urokinase, thus considering the expense involved in its manufacture, its clinical utilization has been restricted. Other examples are Arvin and reptilase. Utilization of these has been restricted for several reasons, but they are still considered as potential replacements for heparin as anticoagulants. Some researchers have noticed that optimum dose plays an important role and is one of the key factors in determining re-clot formation. Thorough investigation is required to overcome any shortcomings and increase the acceptance of these enzymes in therapeutic use [ 47 ].

1.9.2.3. The role of enzymes in digestive disorders and inflammations

Enzymes play an essential role in the management of various digestive disorders, such as exocrine pancreatic insufficiency [ 48 ]. Supplementation with enzymes may also be advantageous for other conditions associated with poor digestion, such as lactose intolerance. Generally, pancreatic enzymes such as porcine and bovine have been the preferred form of supplementation for exocrine pancreatic insufficiency [ 48 ]. Utilization of microbe-derived lipase has presented promise with reports showing benefits alike to pancreatic enzymes, but with a lower dosage concentration and a broader pH range. The safety and efficacy of enzymes derived from microbial species in the treatment of conditions such as malabsorption and lactose intolerance is promising. Plant-derived enzymes, e.g. bromelain from pineapple, serve as active digestive aids in the breakdown of proteins. Synergistic properties have also been reported using a combination of animal-based enzymes and microbe-derived enzymes or bromelain. Buccal administration of pancreatin (derived from an alcoholic extract of animal pancreas) enhances the enzymatic digestion of starch and proteins in patients with pancreatic cysts and pancreatitis. Pancreatin in combination with lipase is used to treat patients with fatty stools. Hydrolytic enzymes such as papain and fungal extracts ( Aspergillus niger and Aspergillus otyzae ) are used to enhance absorption from the small intestine [ 49 ]. These fungal extracts comprise amylases and proteases along with cellulases, which support the breakdown of the otherwise indigestible fibers of cabbages, etc, and thus reduce dyspepsia and flatulence [ 50 ]. Currently, micro-organisms are used at a large scale for the production of therapeutic enzymes. Among various micro-organisms Saccharomyces cerevisiae, Saccharomyces fragilis, Bacillus subtilis and two Aspergillus species are considered safe by the FDA (USA) for obtaining oral β -galactosidase (from A. oryzae ) which is often used by patients suffering from inherited intestinal disease lactose deficiency [ 51 ]. Children with this genetic disorder children are incapable of digesting milk lactose. Enzymatic preparations such as β -galactosidase catalyze the conversion of lactose to glucose and galactose, which are quickly absorbed by the intestine. Other enzymatic preparations, e.g. penicillinase (from B. subtilis ) are often used to treat hypersensitivity reactions caused by the antibiotic penicillin [ 52 ]. This enzyme catalyzes the conversion of penicillin to penicillanic acid, which is non-immunogenic. In addition, microbial and plant hydrolases are also used to decrease inflammation and edema [ 53 ]. Thrombin, trypsin, chymotrypsin, papain, streptokinase, streptodornase and sempeptidase are under clinical trial investigation. These enzymatic preparations are administered orally and have considerable proteolytic activity in the serum. Streptodornase has also displayed pain-relieving action on systemic injection [ 54 ]. Preparations have also been used to clean dirty wounds and necrotic tissue and to remove debris from second and third degree burns.

1.10. Plants and algae enzyme systems

Plant based foods are usually consumed in their raw form [ 68 ]. This eases the main concern with animal-based enzymes by preserving the integrity of the enzymes themselves. Moreover, plant-based digestive enzymes are effective over a broad scope of pH levels. This range is usually between 3.0 and 9.0, which is highly well-matched with the human gastrointestinal environment [ 55 – 72 ]. Thus plant-based enzymes are compatible for supporting comprehensive digestive health. Protease, amylase, lipase and cellulose are the important enzymes and are present in plants. Protease breaks down protein that can be present in meat, fish, poultry, eggs, cheese and nuts. Amylase assists your body with the breakdown and subsequent absorption of carbohydrates and starches. Lipase aids the digestion of fat. When your diet includes lipase-rich foods, it eases the production burden on the gall bladder, liver and pancreas. Cellulase is present in many fruits and vegetables, and it breaks down food fibers, which increases their nutritional value to our bodies. The presence of cellulase in plant-based sources is important, because it is not naturally present in the human body. Fruits and vegetables are an ideal source for enzymes. They are enzyme-rich and easily consumed without needing to be cooked or processed, ultimately preserving the full functionality of the enzymes. By using plant biotechnology several enzymes can be produced from plants as well algal resources [ 56 – 72 ].

During algal photosynthesis various proteins and enzymes are produced which can be utilized in economic development and environment management, such as in wastewater treatment, production of fine chemicals, and biodiesel production [ 56 – 72 ]. Due to their potential to capture and fix carbon dioxide using solar energy, photosynthetic marine algae are considered as potential models for the production of proteins. It has been recently observed that algal chloroplasts can be transformed for the production recombinant proteins [ 55 ]. Five different classes of recombinant enzymes; xylanase, α-galactosidase, phytase, phosphate anhydrolase, and β-mannanase, D. tertiolecta or C. reinhardtii were in the plastids of D. tertiolecta or C. reinhardtii. Similar strategies should allow for recombinant protein production in many species of marine algae [ 55 ].

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Cooper GM. The Cell: A Molecular Approach. 2nd edition. Sunderland (MA): Sinauer Associates; 2000.

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The Cell: A Molecular Approach. 2nd edition.

The central role of enzymes as biological catalysts.

A fundamental task of proteins is to act as enzymes —catalysts that increase the rate of virtually all the chemical reactions within cells. Although RNAs are capable of catalyzing some reactions, most biological reactions are catalyzed by proteins. In the absence of enzymatic catalysis, most biochemical reactions are so slow that they would not occur under the mild conditions of temperature and pressure that are compatible with life. Enzymes accelerate the rates of such reactions by well over a million-fold, so reactions that would take years in the absence of catalysis can occur in fractions of seconds if catalyzed by the appropriate enzyme. Cells contain thousands of different enzymes, and their activities determine which of the many possible chemical reactions actually take place within the cell.

• The Catalytic Activity of Enzymes

Like all other catalysts, enzymes are characterized by two fundamental properties. First, they increase the rate of chemical reactions without themselves being consumed or permanently altered by the reaction. Second, they increase reaction rates without altering the chemical equilibrium between reactants and products.

These principles of enzymatic catalysis are illustrated in the following example, in which a molecule acted upon by an enzyme (referred to as a substrate [ S ]) is converted to a product ( P ) as the result of the reaction. In the absence of the enzyme, the reaction can be written as follows:

The chemical equilibrium between S and P is determined by the laws of thermodynamics (as discussed further in the next section of this chapter) and is represented by the ratio of the forward and reverse reaction rates ( S → P and P → S , respectively). In the presence of the appropriate enzyme, the conversion of S to P is accelerated, but the equilibrium between S and P is unaltered. Therefore, the enzyme must accelerate both the forward and reverse reactions equally. The reaction can be written as follows:

Note that the enzyme ( E ) is not altered by the reaction, so the chemical equilibrium remains unchanged, determined solely by the thermodynamic properties of S and P .

The effect of the enzyme on such a reaction is best illustrated by the energy changes that must occur during the conversion of S to P ( Figure 2.22 ). The equilibrium of the reaction is determined by the final energy states of S and P , which are unaffected by enzymatic catalysis. In order for the reaction to proceed, however, the substrate must first be converted to a higher energy state, called the transition state . The energy required to reach the transition state (the activation energy ) constitutes a barrier to the progress of the reaction, limiting the rate of the reaction. Enzymes (and other catalysts) act by reducing the activation energy , thereby increasing the rate of reaction. The increased rate is the same in both the forward and reverse directions, since both must pass through the same transition state.

Figure 2.22

Energy diagrams for catalyzed and uncatalyzed reactions. The reaction illustrated is the simple conversion of a substrate S to a product P. Because the final energy state of P is lower than that of S , the reaction proceeds from left to right. For the (more...)

The catalytic activity of enzymes involves the binding of their substrates to form an enzyme- substrate complex ( ES ). The substrate binds to a specific region of the enzyme, called the active site . While bound to the active site , the substrate is converted into the product of the reaction, which is then released from the enzyme. The enzyme-catalyzed reaction can thus be written as follows:

Note that E appears unaltered on both sides of the equation, so the equilibrium is unaffected. However, the enzyme provides a surface upon which the reactions converting S to P can occur more readily. This is a result of interactions between the enzyme and substrate that lower the energy of activation and favor formation of the transition state.

• Mechanisms of Enzymatic Catalysis

The binding of a substrate to the active site of an enzyme is a very specific interaction. Active sites are clefts or grooves on the surface of an enzyme, usually composed of amino acids from different parts of the polypeptide chain that are brought together in the tertiary structure of the folded protein. Substrates initially bind to the active site by noncovalent interactions, including hydrogen bonds, ionic bonds, and hydrophobic interactions. Once a substrate is bound to the active site of an enzyme, multiple mechanisms can accelerate its conversion to the product of the reaction.

Although the simple example discussed in the previous section involved only a single substrate molecule, most biochemical reactions involve interactions between two or more different substrates. For example, the formation of a peptide bond involves the joining of two amino acids. For such reactions, the binding of two or more substrates to the active site in the proper position and orientation accelerates the reaction ( Figure 2.23 ). The enzyme provides a template upon which the reactants are brought together and properly oriented to favor the formation of the transition state in which they interact.

Figure 2.23

Enzymatic catalysis of a reaction between two substrates. The enzyme provides a template upon which the two substrates are brought together in the proper position and orientation to react with each other.

Enzymes accelerate reactions also by altering the conformation of their substrates to approach that of the transition state. The simplest model of enzyme- substrate interaction is the lock-and-key model , in which the substrate fits precisely into the active site ( Figure 2.24 ). In many cases, however, the configurations of both the enzyme and substrate are modified by substrate binding—a process called induced fit . In such cases the conformation of the substrate is altered so that it more closely resembles that of the transition state. The stress produced by such distortion of the substrate can further facilitate its conversion to the transition state by weakening critical bonds. Moreover, the transition state is stabilized by its tight binding to the enzyme, thereby lowering the required energy of activation.

Figure 2.24

Models of enzyme-substrate interaction. (A) In the lock-and-key model, the substrate fits precisely into the active site of the enzyme. (B) In the induced-fit model, substrate binding distorts the conformations of both substrate and enzyme. This distortion (more...)

In addition to bringing multiple substrates together and distorting the conformation of substrates to approach the transition state, many enzymes participate directly in the catalytic process. In such cases, specific amino acid side chains in the active site may react with the substrate and form bonds with reaction intermediates. The acidic and basic amino acids are often involved in these catalytic mechanisms, as illustrated in the following discussion of chymotrypsin as an example of enzymatic catalysis.

Chymotrypsin is a member of a family of enzymes (serine proteases) that digest proteins by catalyzing the hydrolysis of peptide bonds. The reaction can be written as follows:

The different members of the serine protease family (including chymotrypsin, trypsin, elastase, and thrombin) have distinct substrate specificities; they preferentially cleave peptide bonds adjacent to different amino acids. For example, whereas chymotrypsin digests bonds adjacent to hydrophobic amino acids, such as tryptophan and phenylalanine, trypsin digests bonds next to basic amino acids, such as lysine and arginine. All the serine proteases, however, are similar in structure and use the same mechanism of catalysis. The active sites of these enzymes contain three critical amino acids—serine, histidine, and aspartate—that drive hydrolysis of the peptide bond . Indeed, these enzymes are called serine proteases because of the central role of the serine residue.

Substrates bind to the serine proteases by insertion of the amino acid adjacent to the cleavage site into a pocket at the active site of the enzyme ( Figure 2.25 ). The nature of this pocket determines the substrate specificity of the different members of the serine protease family. For example, the binding pocket of chymotrypsin contains hydrophobic amino acids that interact with the hydrophobic side chains of its preferred substrates. In contrast, the binding pocket of trypsin contains a negatively charged acidic amino acid (aspartate), which is able to form an ionic bond with the lysine or arginine residues of its substrates.

Figure 2.25

Substrate binding by serine proteases. The amino acid adjacent to the peptide bond to be cleaved is inserted into a pocket at the active site of the enzyme. In chymotrypsin, the pocket binds hydrophobic amino acids; the binding pocket of trypsin contains (more...)

Substrate binding positions the peptide bond to be cleaved adjacent to the active site serine ( Figure 2.26 ). The proton of this serine is then transferred to the active site histidine. The conformation of the active site favors this proton transfer because the histidine interacts with the negatively charged aspartate residue. The serine reacts with the substrate , forming a tetrahedral transition state. The peptide bond is then cleaved, and the C-terminal portion of the substrate is released from the enzyme. However, the N-terminal peptide remains bound to serine. This situation is resolved when a water molecule (the second substrate) enters the active site and reverses the preceding reactions. The proton of the water molecule is transferred to histidine, and its hydroxyl group is transferred to the peptide, forming a second tetrahedral transition state. The proton is then transferred from histidine back to serine, and the peptide is released from the enzyme, completing the reaction.

Figure 2.26

Catalytic mechanism of chymotrypsin. Three amino acids at the active site (Ser-195, His-57, and Asp-102) play critical roles in catalysis.

This example illustrates several features of enzymatic catalysis; the specificity of enzyme- substrate interactions, the positioning of different substrate molecules in the active site , and the involvement of active-site residues in the formation and stabilization of the transition state. Although the thousands of enzymes in cells catalyze many different types of chemical reactions, the same basic principles apply to their operation.

In addition to binding their substrates, the active sites of many enzymes bind other small molecules that participate in catalysis. Prosthetic groups are small molecules bound to proteins in which they play critical functional roles. For example, the oxygen carried by myoglobin and hemoglobin is bound to heme, a prosthetic group of these proteins. In many cases metal ions (such as zinc or iron) are bound to enzymes and play central roles in the catalytic process. In addition, various low-molecular-weight organic molecules participate in specific types of enzymatic reactions. These molecules are called coenzymes because they work together with enzymes to enhance reaction rates. In contrast to substrates, coenzymes are not irreversibly altered by the reactions in which they are involved. Rather, they are recycled and can participate in multiple enzymatic reactions.

Coenzymes serve as carriers of several types of chemical groups. A prominent example of a coenzyme is nicotinamide adenine dinucleotide ( NAD + ), which functions as a carrier of electrons in oxidation-reduction reactions ( Figure 2.27 ). NAD + can accept a hydrogen ion (H + ) and two electrons (e - ) from one substrate , forming NADH. NADH can then donate these electrons to a second substrate, re-forming NAD + . Thus, NAD + transfers electrons from the first substrate (which becomes oxidized) to the second (which becomes reduced).

Figure 2.27

Role of NAD + in oxidation-reduction reactions. (A) Nicotinamide adenine dinucleotide (NAD + ) acts as a carrier of electrons in oxidation-reduction reactions by accepting electrons (e - ) to form NADH. (B) For example, NAD + can accept electrons from one substrate (more...)

Several other coenzymes also act as electron carriers, and still others are involved in the transfer of a variety of additional chemical groups (e.g., carboxyl groups and acyl groups; Table 2.1 ). The same coenzymes function together with a variety of different enzymes to catalyze the transfer of specific chemical groups between a wide range of substrates. Many coenzymes are closely related to vitamins, which contribute part or all of the structure of the coenzyme. Vitamins are not required by bacteria such as E. coli but are necessary components of the diets of human and other higher animals, which have lost the ability to synthesize these compounds.

Examples of Coenzymes and Vitamins.

• Regulation of Enzyme Activity

An important feature of most enzymes is that their activities are not constant but instead can be modulated. That is, the activities of enzymes can be regulated so that they function appropriately to meet the varied physiological needs that may arise during the life of the cell.

One common type of enzyme regulation is feedback inhibition , in which the product of a metabolic pathway inhibits the activity of an enzyme involved in its synthesis. For example, the amino acid isoleucine is synthesized by a series of reactions starting from the amino acid threonine ( Figure 2.28 ). The first step in the pathway is catalyzed by the enzyme threonine deaminase, which is inhibited by isoleucine, the end product of the pathway. Thus, an adequate amount of isoleucine in the cell inhibits threonine deaminase, blocking further synthesis of isoleucine. If the concentration of isoleucine decreases, feedback inhibition is relieved, threonine deaminase is no longer inhibited, and additional isoleucine is synthesized. By so regulating the activity of threonine deaminase, the cell synthesizes the necessary amount of isoleucine but avoids wasting energy on the synthesis of more isoleucine than is needed.

Figure 2.28

Feedback inhibition. The first step in the conversion of threonine to iso-leucine is catalyzed by the enzyme threonine deaminase. The activity of this enzyme is inhibited by isoleucine, the end product of the pathway.

Feedback inhibition is one example of allosteric regulation , in which enzyme activity is controlled by the binding of small molecules to regulatory sites on the enzyme ( Figure 2.29 ). The term “ allosteric regulation ” derives from the fact that the regulatory molecules bind not to the catalytic site, but to a distinct site on the protein ( allo = “other” and steric = “site”). Binding of the regulatory molecule changes the conformation of the protein, which in turn alters the shape of the active site and the catalytic activity of the enzyme. In the case of threonine deaminase, binding of the regulatory molecule (isoleucine) inhibits enzymatic activity. In other cases regulatory molecules serve as activators, stimulating rather than inhibiting their target enzymes .

Figure 2.29

Allosteric regulation. In this example, enzyme activity is inhibited by the binding of a regulatory molecule to an allosteric site. In the absence of inhibitor, the substrate binds to the active site of the enzyme and the reaction proceeds. The binding (more...)

The activities of enzymes can also be regulated by their interactions with other proteins and by covalent modifications, such as the addition of phosphate groups to serine, threonine, or tyrosine residues. Phosphorylation is a particularly common mechanism for regulating enzyme activity; the addition of phosphate groups either stimulates or inhibits the activities of many different enzymes ( Figure 2.30 ). For example, muscle cells respond to epinephrine (adrenaline) by breaking down glycogen into glucose, thereby providing a source of energy for increased muscular activity. The breakdown of glycogen is catalyzed by the enzyme glycogen phosphorylase, which is activated by phosphorylation in response to the binding of epinephrine to a receptor on the surface of the muscle cell. Protein phosphorylation plays a central role in controlling not only metabolic reactions but also many other cellular functions, including cell growth and differentiation.

Figure 2.30

Protein phosphorylation. Some enzymes are regulated by the addition of phosphate groups to the side-chain OH groups of serine (as shown here), threonine, or tyrosine residues. For example, the enzyme glycogen phosphorylase, which catalyzes the conversion (more...)

• Cite this Page Cooper GM. The Cell: A Molecular Approach. 2nd edition. Sunderland (MA): Sinauer Associates; 2000. The Central Role of Enzymes as Biological Catalysts.
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Principles of Biochemistry/Enzymes

Enzymes are organic catalysts that are important to all living things due to the continuous-controlled chemical activities in cells. Enzymes regulate metabolism by altering the rate of chemical reactions. Activation energy is decreased in order to alter chemical reaction rates. In addition, enzymes are able to transform one form of energy to another. Due to the fact that enzymes are highly selective, they may only catalyze one or a specific class of reactions. The place where an enzyme binds onto the substrate is called an active site . A substrate is the molecule that enzyme acts upon.

There are two theories that describe the binding of enzymes: 1) Lock and Key Theory and 2) Induced Fit Theory.

1) Lock and Key Theory: The shape of the enzyme's active site is complementary to that of its substrate

Temperature

Because most enzyme reactions are reversible, an enzyme can synthesize and decompose molecules. Enzymes reaction rate is dependable on several factors: pH, temperature, and concentration of both the enzyme and substrate. Generally, the rate of enzyme reaction would increase as temperature increase; however, if the optimal temperature—usually around 40 ° C-- is reached the enzyme would denatured and loss its ability to react with the substrate.

Despite the fact that every enzymes has their own optimal pH value, most enzyme reactions have higher reaction rates between pH value 6 - 9. The maximal activity rate of enzyme reaction in human body is around pH 7.2, with the exception of pepsin, which has a high reaction rate under acidic condition ( usually around pH 2), and pancreatic enzyme, which has a high reaction rate under alkaline condition ( usually around pH 9)

Concentrations

Figure 2 shows the relationship between concentration and reaction rate. Reference: en.Wikipedia, author: fullofstars

Enzyme Inhibitors There are two categories of enzyme inhibitors: 1) Reversible inhibitors and 2) irreversible inhibitors. Reversible inhibitors are kinetically distinguishable, and is characterized by rapid dissociation of enzyme-inhibitor complex.

There are three sub-categories in reversible inhibitors: 1) Competitive 2) Uncompetitive and 3) Non-competitive Competitive inhibition

Figure 3 demonstrates how competitive inhibition of enzymes work. Reference: en.Wikipedia, Authored by Jerry Crimson Mann, modified by TimVickers, vectorized by Fvasconcellos

Uncompetitive inhibition In an uncompetitive inhibition, inhibitor only binds to enzyme-substrate complex. In addition, in an uncompetitive inhibition, both concentration of substrate and maximum velocity decrease.

Noncompetitive inhibition

A noncompetitive inhibition reaction is nonreversible due to the act that strong covalent bonding, that cannot be displaced by the addition of excess substrate, between a molecule and a enzyme. That molecule is also called a noncompetitive inhibitor. In non-competitive inhibition, inhibitor and substrate can bind to enzyme simultaneously at different binding sites. Furthermore, it can bind free enzyme or enzyme substrate complex. Also, during a noncompetitive inhibition, concentration of substrate will increase while maximum velocity will decrease.

There are three sub-categories of irreversible inhibitors: 1) group specific, 2) reactive substrate analogs, 3) mechanism-based inhibitors. Group Specific inhibitors only react with specific side chains of amino acid or with certain chemical groups.

Reactive substrate analogs, also known as affinity label. In reactive substrate analogs, molecules that are structurally similar to the substrate for an enzyme and that covalently bind to active-site residue.

Mechanism-based inhibitors is also known as suicide inhibitors. There are three steps in the mechanism, the first one is oxidation which is then followed by alkylation and lastly by protonation.

Cofactors are needed for some catalytic reactions. Cofactors can either be inorganic elements (metals) or complex organic molecules, in which many of them are derived from vitamins. A holoenzyme is a catalytically activated enzyme; while an apoenzyme is an enzyme without cofactor.

Gibbs Free Energy (ΔG)

Enzymes decrease activation energy. Enzymes accelerates reactions by lowering Gibbs free energy at transition state, which is also known as the free energy of activation. Gibbs free energy also provides information about spontaneity of a reaction.

Reaction is spontaneous if : ΔG<0

Reaction is not spontaneous if : ΔG > 0

Reaction is at equilibrium if : ΔG=0

To determine the total Gibbs free energy of a reaction, take the summation of Gibbs free energy of formation of products and subtract the sum of Gibbs free energy of formation of reactants from it. Another important point regarding ΔG is that ΔG does not explain the reaction rate of a reaction.

An active site of an enzyme is where substrate bind to and is also where chemical reaction take place. An active site is a small part on a protein and is cleft, or crevice. It also has a unique microenvironment and substrates are bind by several weak interactions. furthermore, specificity of the enzyme depends on the precise arrangement of atoms.

Biochemical Reactions

There are two main enzymatic reactions: 1) Sequential Reactions and 2) Double Displacement Reaction

1) Sequential Reaction: In sequential reactions, all substrates are bind to the enzyme before any product is released. In an ordered sequential reaction, substrates bind to enzyme in a defined sequence; while in a random sequential reaction, substrates bind to enzyme in an undefined sequence.

2) Double Displacement Reaction: In double displacement reactions, one or more products are released before all substrates bind to the enzyme.

[1]

References [ edit | edit source ]

• ↑ Kaplan PCAT 2010-2011 Edition

• Book:Principles of Biochemistry

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18.6: Enzyme Action

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  Learning Objectives

• To describe the interaction between an enzyme and its substrate.

Enzyme-catalyzed reactions occur in at least two steps. In the first step, an enzyme molecule (E) and the substrate molecule or molecules (S) collide and react to form an intermediate compound called the enzyme-substrate (E–S) complex . (This step is reversible because the complex can break apart into the original substrate or substrates and the free enzyme.) Once the E–S complex forms, the enzyme is able to catalyze the formation of product (P), which is then released from the enzyme surface:

\[S + E \rightarrow E–S \tag{\(\PageIndex{1}\)} \]

\[E–S \rightarrow P + E \tag{\(\PageIndex{2}\)} \]

Hydrogen bonding and other electrostatic interactions hold the enzyme and substrate together in the complex. The structural features or functional groups on the enzyme that participate in these interactions are located in a cleft or pocket on the enzyme surface. This pocket, where the enzyme combines with the substrate and transforms the substrate to product is called the active site of the enzyme (Figure \(\PageIndex{1}\)).

The active site of an enzyme possesses a unique conformation (including correctly positioned bonding groups) that is complementary to the structure of the substrate, so that the enzyme and substrate molecules fit together in much the same manner as a key fits into a tumbler lock. In fact, an early model describing the formation of the enzyme-substrate complex was called the lock-and-key model (Figure \(\PageIndex{2}\)). This model portrayed the enzyme as conformationally rigid and able to bond only to substrates that exactly fit the active site.

Working out the precise three-dimensional structures of numerous enzymes has enabled chemists to refine the original lock-and-key model of enzyme actions. They discovered that the binding of a substrate often leads to a large conformational change in the enzyme, as well as to changes in the structure of the substrate or substrates. The current theory, known as the induced-fit model, says that enzymes can undergo a change in conformation when they bind substrate molecules, and the active site has a shape complementary to that of the substrate only after the substrate is bound, as shown for hexokinase in Figure \(\PageIndex{3}\). After catalysis, the enzyme resumes its original structure.

The structural changes that occur when an enzyme and a substrate join together bring specific parts of a substrate into alignment with specific parts of the enzyme’s active site. Amino acid side chains in or near the binding site can then act as acid or base catalysts, provide binding sites for the transfer of functional groups from one substrate to another or aid in the rearrangement of a substrate. The participating amino acids, which are usually widely separated in the primary sequence of the protein, are brought close together in the active site as a result of the folding and bending of the polypeptide chain or chains when the protein acquires its tertiary and quaternary structure. Binding to enzymes brings reactants close to each other and aligns them properly, which has the same effect as increasing the concentration of the reacting compounds.

Example \(\PageIndex{1}\)

• What type of interaction would occur between an OH group present on a substrate molecule and a functional group in the active site of an enzyme?
• Suggest an amino acid whose side chain might be in the active site of an enzyme and form the type of interaction you just identified.
• An OH group would most likely engage in hydrogen bonding with an appropriate functional group present in the active site of an enzyme.
• Several amino acid side chains would be able to engage in hydrogen bonding with an OH group. One example would be asparagine, which has an amide functional group.

Exercise \(\PageIndex{1}\)

• What type of interaction would occur between an COO − group present on a substrate molecule and a functional group in the active site of an enzyme?

One characteristic that distinguishes an enzyme from all other types of catalysts is its substrate specificity . An inorganic acid such as sulfuric acid can be used to increase the reaction rates of many different reactions, such as the hydrolysis of disaccharides, polysaccharides, lipids, and proteins, with complete impartiality. In contrast, enzymes are much more specific. Some enzymes act on a single substrate, while other enzymes act on any of a group of related molecules containing a similar functional group or chemical bond. Some enzymes even distinguish between D- and L-stereoisomers, binding one stereoisomer but not the other. Urease, for example, is an enzyme that catalyzes the hydrolysis of a single substrate—urea—but not the closely related compounds methyl urea, thiourea, or biuret. The enzyme carboxypeptidase, on the other hand, is far less specific. It catalyzes the removal of nearly any amino acid from the carboxyl end of any peptide or protein.

Enzyme specificity results from the uniqueness of the active site in each different enzyme because of the identity, charge, and spatial orientation of the functional groups located there. It regulates cell chemistry so that the proper reactions occur in the proper place at the proper time. Clearly, it is crucial to the proper functioning of the living cell.

A substrate binds to a specific region on an enzyme known as the active site, where the substrate can be converted to product. The substrate binds to the enzyme primarily through hydrogen bonding and other electrostatic interactions. The induced-fit model says that an enzyme can undergo a conformational change when binding a substrate. Enzymes exhibit varying degrees of substrate specificity.

What are enzymes and what do they do in our bodies? Enzymes are basically proteins that are produced by living organisms to bring about certain metabolic and biochemical reactions in the body. They are biological catalysts that speed up reactions inside the body. Let’s find out more about them.

Suggested Videos:

What is the structure of enzymes.

Enzymes, as mentioned above, are biological catalysts. While they hasten or speed up a process, they are actually providing an alternative pathway for the process. But, in the process, the structure or composition of the enzymes remain unaltered.

Enzymes are actually made up of 1000s of amino acids that are linked in a  specific way to form different enzymes. The enzyme chains fold over to form unique shapes and it is these shapes that provide the enzyme with its characteristic chemical potential. Most enzymes also contain a non-protein component known as the co-facto r.   

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Types of Enzymes:

The biochemical reactions occurring in the body are basically of 6 types and the enzymes that bring about these reactions are named accordingly:

• Oxidoreductases: These enzymes bring about oxidation and reduction reactions and hence are called oxidoreductases. In these reactions, electrons in the form of hydride ions or hydrogen atoms are transferred. When a substrate is being oxidized, these enzymes act as the hydrogen donor. These enzymes are called dehydrogenases or reductases. When the oxygen atom is the acceptor, these enzymes are called oxidases.
• Transferases: These enzymes are responsible for transferring functional groups from one molecule to another. Example: alanine aminotransferase which shuffles the alpha‐amino group between alanine and aspartate etc. Some transferases also transfer phosphate groups between ATP and other compounds, sugar residues to form disaccharides such as hexokinase in glycolysis.
• Hydrolases: These enzymes catalyze reactions that involve the process of hydrolysis.They break single bonds by adding water. Some hydrolases function as digestive enzymes because they break the peptide bonds in proteins. Hydrolases can also be a type of transferases as they transfer the water molecule from one compound to another. Example: Glucose-6-phosphatase that removes the phosphate group from glucose-6-phosphate, leaving glucose and H 3 PO 4 .
• Lyases:   These enzymes catalyze reactions where functional groups are added to break double bonds in molecules or where double bonds are formed by the removal of functional groups. Example: Pyruvate decarboxylase is a lyase that removes CO 2  from pyruvate. Other examples include deaminases and dehydratases.
• Isomerases: These enzymes catalyze the reactions where a functional group is moved to another position within the same molecule such that the resulting molecule is actually an isomer of the earlier molecule. Example: triosephosphate isomerase and phosphoglucose isomerase for converting glucose 6-phosphate to fructose 6-phosphate.
• Ligases: These enzymes perform a function that is opposite to that of the hydrolases. Where hydrolases break bonds by adding water, ligases form bonds by removal of the water component. There are different subclasses of ligases which involve the synthesis of ATP.

How do enzymes work?

For any reaction to occur in the universe, there is an energy requirement. In cases where there is no activation energy provided, a catalyst plays an important role to reduce the activation energy and carried forward the reaction. This works in animals and plants as well. Enzymes help reduce the activation energy of the complex molecules in the reaction.  The following steps simplify how an enzyme works to speed up a reaction:

Step 1 : Each enzyme has an ‘active site’ which is where one of the substrate molecules can bind to. Thus, an enzyme- substrate complex is formed.

Step 2: This enzyme-substrate molecule now reacts with the second substrate to form the product and the enzyme is liberated as the second product.

There are many theories that explain how enzymes work. But, there are two important theories that we will discuss here.

Theory 1: Lock and Key Hypothesis

This is the most accepted of the theories of enzyme action.

This theory states that the substrate fits exactly into the active site of the enzyme to form an enzyme-substrate complex. This model also describes why enzymes are so specific in their action because they are specific to the substrate molecules.

Theory 2: Induced Fit Hypothesis

This is similar to the lock and key hypothesis. It says that the shape of the enzyme molecule changes as it gets closer to the substrate molecule in such a way that the substrate molecule fits exactly into the active site of the enzyme.

What factors affect enzyme activity in the cell?

• Concentration of Enzymes and Substrates: The rate of reaction increases with increasing substrate concentration up to a point, beyond which any further increase in substrate concentration produces no significant change in reaction rate. This occurs because after a certain concentration of the substrate, all the active sites on the enzyme are full and no further reaction can occur.
• Temperature: With the increase in temperature, the enzyme activity increases because of the increase in kinetic energy of the molecules. There is an optimum level when the enzymes work at the best and maximum. This temperature is often the normal body temperature of the body. When the temperature increases beyond a certain limit, enzymes, which are actually made up of proteins, begin to disintegrate and the rate of reaction slows down.
• pH: Enzymes are very sensitive to changes in the pH and work in a very small window of permissible pH levels. Below or above the optimum pH level, there is a risk of the enzymes disintegrating and thereby the reaction slows down.
• Inhibitors: Presence of certain substances that inhibit the action of a particular enzyme. This occurs when the inhibiting substance attaches itself to the active site of the enzyme thereby preventing the substrate attachment and slows down the process.

Solved Example for You

Q: An enzyme acts by?

a. Increasing the energy of activation

b. Decreasing the energy of activation

c. Decreasing the pH

d. Increasing the pH

Sol: a. Increasing the energy of activation

The reactants do not undergo chemical change automatically. They do so in the transition state. Transition state has more free energy than reactants or products. The inability of reactants to undergo change due to the requirement of extra energy for converting them to transition state is called as ‘Energy Barrier’. The energy required to overcome energy barrier is called as ‘Activation Energy’.

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Enzymes: How they work and what they do

Enzymes help with specific functions that are vital to the operation and overall health of the body. They help speed up chemical reactions in the human body. They are essential for respiration, digesting food, muscle and nerve function, and more.

Each cell in the human body contains thousands of enzymes. Enzymes provide help with facilitating chemical reactions within each cell.

Since they are not destroyed during the process, a cell can reuse each enzyme repeatedly.

This article reviews what enzymes are and the roles they play in various parts of the body.

The majority of enzymes are proteins, though some are Ribonucleic acid (RNA) molecules. RNA molecules translate information from DNA and create proteins.

Each cell contains thousands of enzymes, providing specific help throughout the body.

Enzymes help with the chemical reactions that keep a person alive and well. For example, they perform a necessary function for metabolism, the process of breaking down food and drink into energy.

Enzymes speed up (catalyze) chemical reactions in cells. More specifically, they lower the threshold necessary to start the intended reaction. They do this by binding to another substance known as a substrate.

What do enzymes do?

Enzymes provide support for many important processes within the body. Some examples include:

• The digestive system: Enzymes help the body break down larger complex molecules into smaller molecules, such as glucose, so that the body can use them as fuel.
• DNA replication: Each cell in the body contains DNA. Each time a cell divides, the cell needs to copy its DNA. Enzymes help in this process by unwinding the DNA coils.
• Liver enzymes: The liver breaks down toxins in the body. To do this, it uses a range of enzymes the facilitate the process of destroying the toxins.

Other activities enzymes help with include :

• hormone production
• cell regulation
• creating movement to make the muscle contract
• transporting materials around a cell
• respiration
• signal transduction

How enzymes work

The “lock and key” model was first proposed in 1894. In this model, an enzyme’s active site is a specific shape, and only the substrate will fit into it, like a lock and key.

A newer model, the induced-fit model, helps to account for reactions between substrates and active sites that are not exact fits.

In this model, the active site changes shape as it interacts with the substrate. Once the substrate fully locks in and in the exact position, the catalysis can begin.

The perfect conditions

Enzymes can only work in certain conditions. Most enzymes in the human body work best at around 98.6-degrees Fahrenheit (F) (37°C), which is the body’s typical temperature. At lower temperatures, they may still work but much more slowly.

If the temperature is too high or if the environment is too acidic or alkaline, the enzyme changes shape; this alters the shape of the active site so that substrates cannot bind to it. This is denaturing.

Different enzymes tolerate different levels of acidity. For instance, enzymes in the intestines work best at around 8 pH , whereas enzymes in the stomach work best at about pH 1.5 because the stomach is much more acidic.

Some enzymes cannot function unless they attach to a specific non-protein molecule, known as cofactors. There are two types of cofactors, ions and coenzymes.

Ions are inorganic molecules that loosely bond to the enzyme to ensure it can function. By contrast, coenzymes are organic molecules that also loosely bond with and allow an enzyme to do its job.

When a cofactor bonds tightly with an enzyme, it is known as a prosthetic group.

To ensure that the body’s systems work correctly, it is sometimes necessary to slow down enzyme function. For instance, if an enzyme makes too much of a product, then the body needs a way to reduce or stop the production.

Several factors can limit enzyme activity levels, including:

• Competitive inhibitors: This inhibitor molecule blocks the active site so that the substrate has to compete with the inhibitor to attach to the enzyme.
• Non-competitive inhibitors: This molecule binds to an enzyme somewhere other than the active site and reduces how effectively it works.
• Uncompetitive inhibitors: This inhibitor binds to the enzyme and substrate. The products leave the active site less easily, which slows the reaction.
• Irreversible inhibitors: This is an irreversible inhibitor, which binds to an enzyme and permanently inactivates it.

Examples of specific enzymes

Thousands of enzymes in the human body exist to perform around 5,000 different functions. A few examples include:

• Lipases: This group of enzymes help digest fats in the gut.
• Amylase: In the saliva, amylase helps change starches into sugars.
• Maltase: This also occurs in the saliva, and breaks the sugar maltose into glucose.
• Trypsin: These enzymes break proteins down into amino acids in the small intestine.
• Lactase: Lactase breaks lactose, the sugar in milk, into glucose and galactose.
• Acetylcholinesterase: These enzymes break down the neurotransmitter acetylcholine in nerves and muscles.
• Helicase: Helicase enzymes unravel DNA.
• DNA polymerase: These enzymes synthesize DNA from deoxyribonucleotides.

Types of enzymes

Experts break enzymes down into several different types based on the functions they perform in the body. The different types include :

• oxidoreductases
• transferases

The body needs all of the different types to function properly.

Enzymes play a large part in the day-to-day running of the human body. Enzymes work by combining with molecules to start a chemical reaction. They work best at certain pH levels and temperatures.

They play a vital role in the proper functioning of the digestive system, the nervous system, muscles, and more.

Last medically reviewed on July 8, 2022

• Biology / Biochemistry
• GastroIntestinal / Gastroenterology

How we reviewed this article:

• 1.18: Enzyme function. (2021). https://bio.libretexts.org/Bookshelves/Introductory_and_General_Biology/Book%3A_Introductory_Biology_(CK-12)/01%3A_Introduction_to_Biology/1.18%3A_Enzyme_Function
• Enzyme. (2022). https://www.genome.gov/genetics-glossary/Enzyme
• Enzymes in the body. (n.d.). https://corneotherapy.org/articles/enzymes-in-the-body
• Krise, K. M. (n.d.). Enzymes: Moving at the speed of life. https://www.acs.org/content/acs/en/education/outreach/celebrating-chemistry-editions/2021-ncw/enzymes.html
• Robinson, P. K. (2015). Enzymes: principles and biotechnological applications. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4692135/
• What is dna replication? (2021). https://www.yourgenome.org/facts/what-is-dna-replication

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1.18: Enzyme Function

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As you view Enzyme Animation, focus on this concept: how enzymes function.

The reaction represented by this graph is a combustion reaction involving the reactants glucose (C 6 H 12 O 6 ) and oxygen (O 2 ). The products of the reaction are carbon dioxide (CO 2 ) and water (H 2 O). Energy is also released during the reaction. The enzyme speeds up the reaction by lowering the activation energy needed for the reaction to start. Compare the activation energy with and without the enzyme.

Enzymes generally lower activation energy by reducing the energy needed for reactants to come together and react. For example:

• Enzymes bring reactants together so they don’t have to expend energy moving about until they collide at random. Enzymes bind both reactant molecules (called the substrate ), tightly and specifically, at a site on the enzyme molecule called the active site ( Figure below ).
• By binding reactants at the active site, enzymes also position reactants correctly, so they do not have to overcome intermolecular forces that would otherwise push them apart. This allows the molecules to interact with less energy.
• Enzymes may also allow reactions to occur by different pathways that have lower activation energy.

The active site is specific for the reactants of the biochemical reaction the enzyme catalyzes. Similar to puzzle pieces fitting together, the active site can only bind certain substrates.

This enzyme molecule binds reactant molecules—called substrate—at its active site, forming an enzyme-substrate complex. This brings the reactants together and positions them correctly so the reaction can occur. After the reaction, the products are released from the enzyme’s active site. This frees up the enzyme so it can catalyze additional reactions.

The activities of enzymes also depend on the temperature, ionic conditions, and the pH of the surroundings. Some enzymes work best at acidic pHs, while others work best in neutral environments.

• Digestive enzymes secreted in the acidic environment (low pH) of the stomach help break down proteins into smaller molecules. The main digestive enzyme in the stomach is pepsin , which works best at a pH of about 1.5. These enzymes would not work optimally at other pHs. Trypsin is another enzyme in the digestive system, which breaks protein chains in food into smaller parts. Trypsin works in the small intestine, which is not an acidic environment. Trypsin's optimum pH is about 8.
• Biochemical reactions are optimal at physiological temperatures. For example, mostbiochemical reactions work best at the normal body temperature of 98.6˚F. Many enzymes lose function at lower and higher temperatures. At higher temperatures, an enzyme’s shape deteriorates. Only when the temperature comes back to normal does the enzyme regain its shape and normal activity.
• Enzymes work by lowering the activation energy needed to start biochemical reactions.
• The activities of enzymes depend on the temperature, ionic conditions, and the pH of the surroundings.

Explore More

Explore more i.

Use this resource to answer the questions that follow.

• Enzymes Make the World Go 'Round at http ://www.chem4kids.com/files/bio_enzymes.html .
• What are enzymes?
• How many jobs in the cell can one enzyme do?
• What is the substrate?
• How does an enzyme interact with a substrate?
• List four factors that can regulate enzyme activity?

Explore More II

• Enzyme Kinetics at http://www.kscience.co.uk/animations/model.swf .
• How do enzymes speed up biochemical reactions?
• Where is the active site located? Explain the role of the active site?
• Complete this sentence: The activities of enzymes depends on the __________, __________ conditions, and the __________ of the surroundings.
• Distinguish between the conditions needed for the proper functioning of pepsin and trypsin.

Lock-and-key model

strong>Lock-and-key model n., [lɑk ænd ki ˈmɑdl̩] Definition: a model for enzyme-substrate interaction

Table of Contents

Lock-and-key model Definition

Lock-and-key model is a model for enzyme-substrate interaction suggesting that the enzyme and the substrate possess specific complementary geometric shapes that fit exactly into one another. In this model, enzymes are depicted as highly specific. They must bind to specific substrates before they catalyze chemical reactions . The term is a pivotal concept in enzymology to elucidate the intricate interaction between enzymes and substrates at the molecular level. In the lock-and-key model, the enzyme-substrate interaction suggests that the enzyme and the substrate possess specific complementary geometric shapes that fit exactly into one another. Like a key  into a  lock , only the correct size and shape of the substrate ( the key ) would fit into the  active site  ( the keyhole ) of the enzyme ( the lock ).

Compare: Induced fit model   See also: enzyme , active site , substrate

Lock-and-key vs. Induced Fit Model

At present, two models attempt to explain enzyme-substrate specificity; one of which is the lock-and-key model , and the other is the Induced fit model . The lock and key model theory was first postulated by  Emil Fischer   in 1894. The lock-and-key enzyme action proposes the high specificity of enzymes. However, it does not explain the stabilization of the transition state that the enzymes achieve. The induced fit model (proposed by Daniel Koshland in 1958) suggests that the active site continues to change until the substrate is completely bound to the active site of the enzyme, at which point the final shape and charge are determined. Unlike the lock-and-key model, the induced fit model shows that enzymes are rather flexible structures. Nevertheless, Fischer’s Lock and Key theory laid an important foundation for subsequent research, such as during the refinement of the enzyme-substrate complex mechanism, as ascribed in the induced fit model. The lock-and-key hypothesis has opened ideas where enzyme action is not merely catalytic but incorporates a rather complex process in how they interact with the correct substrates with precision.

Key Components

Components of the lock and key model:

• Enzyme : the enzyme structure is a three-dimensional protein configuration, with an active site from where the substrate binds.
• Substrate : often an organic molecule, a substrate possesses a structural feature that complements the geometry of the enzyme’s active site.

In the lock and key model, both the enzymes and the substrates facilitate the formation of a complex that lowers the activation energy needed for a chemical transformation to occur. Such reduction in the activation energy allows the chemical reaction to proceed at a relatively faster rate, making enzymes crucial in various biological and molecular processes.

Lock-and-key Model Examples

Some of the common examples that are often discussed in the context of the Lock and Key Model are as follows:

• Enzyme lactate dehydrogenase with a specific active site for its substrates, pyruvate and lactate. The complex facilitates the interconversion of pyruvate and lactate during anaerobic respiration
• Enzyme carbonic anhydrase with a specific active site for the substrates carbon dioxide and water. The complex facilitates the hydration of carbon dioxide, forming bicarbonate
• Enzyme lysozyme binding with a bacterial cell wall peptidoglycan, which is a vital immune function

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• Aryal, S. and Karki, P. (2023).  “Lock and Key Model- Mode of Action of Enzymes”. Microbenotes.com. https://microbenotes.com/lock-and-key-model-mode-of-action-of-enzymes/
• Farhana, A., & Lappin, S. L. (2023, May).  Biochemistry, Lactate Dehydrogenase . Nih.gov; StatPearls Publishing. https://www.ncbi.nlm.nih.gov/books/NBK557536/

©BiologyOnline.com. Content provided and moderated by Biology Online Editors.

Last updated on January 11th, 2024

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• Biology Article

The human body is composed of different types of cells, tissues and other complex organs. For efficient functioning, our body releases some chemicals to accelerate biological processes such as respiration, digestion, excretion and a few other metabolic activities to sustain a healthy life. Hence, enzymes are pivotal in all living entities which govern all the biological processes.

Table of Contents

• Explanation
• Classification
• Interactions

Let us understand what are enzymes, types, their structure, mechanism and various factors that affect its activity.

What Are Enzymes?

“Enzymes can be defined as biological polymers that catalyze biochemical reactions.” 

The majority of enzymes are proteins with catalytic capabilities crucial to perform different processes. Metabolic processes and other chemical reactions in the cell are carried out by a set of enzymes that are necessary to sustain life.

The initial stage of metabolic process depends upon the enzymes, which react with a molecule and is called the substrate. Enzymes convert the substrates into other distinct molecules, which are known as products.

The regulation of enzymes has been a key element in clinical diagnosis because of their role in maintaining life processes. The macromolecular components of all enzymes consist of protein, except in the class of RNA catalysts called ribozymes. The word ribozyme is derived from the ribonucleic acid enzyme. Many ribozymes are molecules of ribonucleic acid, which catalyze reactions in one of their own bonds or among other RNAs.

Enzymes are found in all tissues and fluids of the body. Catalysis of all reactions taking place in metabolic pathways is carried out by intracellular enzymes. The enzymes in the plasma membrane govern the catalysis in the cells as a  response to cellular signals and enzymes in the circulatory system regulate the clotting of blood. Most of the critical life processes are established on the functions of enzymes.

Enzyme Structure

Enzymes are a linear chain of amino acids, which give rise to a three-dimensional structure. The sequence of amino acids specifies the structure, which in turn identifies the catalytic activity of the enzyme. Upon heating, the enzyme’s structure denatures, resulting in a loss of enzyme activity, which typically is associated with temperature.

Compared to its substrates, enzymes are typically large with varying sizes, ranging from 62 amino acid residues to an average of 2500 residues found in fatty acid synthase. Only a small section of the structure is involved in catalysis and is situated next to the binding sites. The catalytic site and binding site together constitute the enzyme’s active site. A small number of ribozymes exist which serve as an RNA-based biological catalyst. It reacts in complex with proteins.

Also read:  Amino acids

Enzymes Classification

Earlier, enzymes were assigned names based on the one who discovered them. With further research, classification became more comprehensive.

According to the International Union of Biochemists (I U B), enzymes are divided into six functional classes and are classified based on the type of reaction in which they are used to catalyze. The six kinds of enzymes are hydrolases, oxidoreductases, lyases, transferases, ligases and isomerases.

Listed below is the classification of enzymes discussed in detail:

• Oxidoreductases

These catalyze oxidation and reduction reactions, e.g. pyruvate dehydrogenase, catalysing the oxidation of pyruvate to acetyl coenzyme A.

• Transferases

These catalyze transferring of the chemical group from one to another compound. An example is a transaminase, which transfers an amino group from one molecule to another.

They catalyze the hydrolysis of a bond. For example, the enzyme pepsin hydrolyzes peptide bonds in  proteins .

These catalyze the breakage of bonds without catalysis, e.g. aldolase (an enzyme in glycolysis) catalyzes the splitting of fructose-1, 6-bisphosphate to glyceraldehyde-3-phosphate and dihydroxyacetone phosphate.

They catalyze the formation of an isomer of a compound. Example: phosphoglucomutase catalyzes the conversion of glucose-1-phosphate to glucose-6-phosphate (phosphate group is transferred from one to another position in the same compound) in glycogenolysis (glycogen is converted to glucose for energy to be released quickly).

Ligases catalyze the association of two molecules. For example, DNA ligase catalyzes the joining of two fragments of DNA by forming a phosphodiester bond.

Cofactors are non-proteinous substances that associate with enzymes. A cofactor is essential for the functioning of an enzyme. The protein part of enzymes in cofactors is apoenzyme. An enzyme and its cofactor together constitute the holoenzyme.

There are three kinds of cofactors present in enzymes:

• Prosthetic groups : These are cofactors tightly bound to an enzyme at all times. FAD (flavin adenine dinucleotide) is a prosthetic group present in many enzymes.
• Coenzyme : A coenzyme binds to an enzyme only during catalysis. At all other times, it is detached from the enzyme. NAD is a common coenzyme.
• Metal ions : For the catalysis of certain enzymes, a metal ion is required at the active site to form coordinate bonds. Zinc is a metal ion cofactor used by a number of enzymes.

Examples of Enzymes

Following are some of the examples of enzymes:

Alcoholic beverages generated by fermentation vary a lot based on many factors. Based on the type of the plant’s product, which is to be used and the type of enzyme applied, the fermented product varies.

For example, grapes, honey, hops, wheat, cassava roots, and potatoes depending upon the materials available. Beer, wines and other drinks are produced from plant fermentation.

Food Products

Bread can be considered as the finest example of fermentation in our everyday life.

A small proportion of yeast and sugar is mixed with the batter for making bread. Then one can observe that the bread gets puffed up as a result of fermentation of the sugar by the enzyme action in yeast, which leads to the formation of carbon dioxide gas. This process gives the texture to the bread, which would be missing in the absence of the fermentation process.

Drug Action

Enzyme action can be inhibited or promoted by the use of drugs which tend to work around the active sites of enzymes.

Also Read:   Digestive Enzymes

Mechanism of Enzyme Reaction

Any two molecules have to collide for the reaction to occur along with the right orientation and a sufficient amount of energy. The energy between these molecules needs to overcome the barrier in the reaction. This energy is called activation energy.

Enzymes are said to possess an active site. The active site is a part of the molecule that has a definite shape and the functional group for the binding of reactant molecules. The molecule that binds to the enzyme is referred to as the substrate group. The substrate and the enzyme form an intermediate reaction with low activation energy without any catalysts.

\(\begin{array}{l}reactant(1) + reactant(2) \rightarrow product\\ reactant(1) + enzyme \rightarrow intermediate\\ intermediate + reactant(2) \rightarrow product + enzyme\end{array} \)

The basic mechanism of enzyme action is to catalyze the chemical reactions, which begins with the binding of the substrate with the active site of the enzyme. This active site is a specific area that combines with the substrate.

Enzyme-Substrate Interactions

Enzymes are biocatalysts, which are high molecular weight proteinous compounds. It enhances the reactions which occur in the body during various life processes . It helps the substrate by providing the surface for the reaction to occur. The enzyme comprises hollow spaces occupying groups such as -SH, -COOH, and others on the outer surface. The substrate which has an opposite charge of the enzyme fits into these spaces, just like a key fits into a lock. This substrate binding site is called the active site of an enzyme (E).

The favourable model of enzyme-substrate interaction is called the induced-fit model. This model states that the interaction between substrate and enzyme is weak, and these weak interactions induce conformational changes rapidly and strengthen binding and bring catalytic sites close enough to substrate bonds.

There are four possible major mechanisms of catalysis:

Catalysis by Bond Strain

The induced structural rearrangements in this type of catalysis produce strained substrate bonds that attain transition state more easily. The new conformation forces substrate atoms and catalytic groups like aspartate into conformations that strain substrate bonds.

Covalent Catalysis

The substrate is oriented to active place on the enzymes in such a manner that a covalent intermediate develops between the enzyme and the substrate, in catalysis that occurs by covalent mechanisms. The best example of this involves proteolysis by serine proteases that have both digestive enzymes and various enzymes of the blood clotting cascade. These proteases possess an active site serine whose R group hydroxyl generates a covalent bond with a carbonyl carbon of a peptide bond and results in the hydrolysis of the peptide bond.

Catalysis Involving Acids and Bases

Other mechanisms add to the completion of catalytic events which are launched by strain mechanisms such as the usage of glutamate as a general acid catalyst.

Catalysis by Orientation and Proximity

Enzyme-substrate interactions induce reactive groups into proximity with one another. Also, groups like aspartate are chemically reactive, and their proximity towards the substrate favours their involvement in catalysis.

Action and Nature of Enzymes

Once substrate (S) binds to this active site, they form a complex (intermediate-ES) which then produces the product (P) and the enzyme (E). The substrate which gets attached to the enzyme has a specific structure and that can only fit in a particular enzyme. Hence, by providing a surface for the substrate, an enzyme slows down the activation energy of the reaction. The intermediate state where the substrate binds to the enzyme is called the transition state. By breaking and making the bonds, the substrate binds to the enzyme (remains unchanged), which converts into the product and later splits into product and enzyme. The free enzymes then bind to other substrates and the catalytic cycle continues until the reaction completes.

The enzyme action basically happens in two steps:

Step1:  Combining of enzyme and the reactant/substrate.

  Step 2:  Disintegration of the complex molecule to give the product.

Thus, the whole catalyst action of enzymes is summarized as:

E + S →  [ES] →  [ EP] → E  + P

Biological Catalysts

Catalysts are the substances which play a significant role in the chemical reaction. Catalysis is the phenomenon by which the rate of a chemical reaction is altered/ enhanced without changing themselves. During a chemical reaction, a catalyst remains unchanged, both in terms of quantity and chemical properties. An enzyme is one such catalyst which is commonly known as the biological catalyst.  Enzymes present in the living organisms enhance the rate of reactions which take place within the body.

Biological catalysts, enzymes, are extremely specific that catalyze a single chemical reaction or some closely associated reactions. An enzyme’s exact structure and its active site decide an enzyme’s specificity. Substrate molecules attach themselves at the active site of an enzyme. Initially, substrates associate themselves by noncovalent interactions to the enzymes which include ionic, hydrogen bonds and hydrophobic interactions. Enzymes reduce the reactions and activation energy to progress towards equilibrium quicker than the reactions that are not catalyzed. Both eukaryotic and prokaryotic cells usually make use of allosteric regulation to respond to fluctuations in the state inside the cells.

The nature of enzyme action and factors affecting the enzyme activity are discussed below.

Factors Affecting Enzyme Activity

The conditions of the reaction have a great impact on the activity of the enzymes. Enzymes are particular about the optimum conditions provided for the reactions such as temperature, pH, alteration in substrate concentration, etc.

Typically, enzyme activities are accelerated with increasing temperatures. As enzymes are functional in cells, the feasible conditions for nearly all enzymes are temperatures that are moderate. At higher temperatures, given a specific point, there is a drastic decrease in the activity with the denaturation of enzymes. In diluted solutions, purified enzymes denature quickly compared to enzymes in crude extracts. Denaturation of enzymes can also take place when enzymes are incubated for long durations. More appropriate is to utilize a shorter time duration when it comes to incubation time to gauge the starting velocities of such enzyme reactions.

The International Union of Biochemistry suggests the standard assay temperature to be 30 °C. Almost all enzymes are extremely sensitive to pH change. Just some enzymes feasibly operate with pH above 9 and below 5. Most enzymes have their pH – optimum near to neutrality. Any alteration of pH causes the ionic state of amino acid residues to change in the whole protein and in the active site. The modifications in the ionic state can modify catalysis and substrate binding. The preference of substrate concentration is critical as at lower concentrations, the rate is driven by concentration, however, at high concentrations, the rate does not depend on any increase in the concentration of the substrate.

Active site

Enzymatic catalysis depends upon the activity of amino acid side chains assembled in the active centre. Enzymes bind the substrate into a region of the active site in an intermediate conformation.

Often, the active site is a cleft or a pocket produced by the amino acids which take part in catalysis and substrate binding. Amino acids forming an enzyme’s active site is not contiguous to the other along the sequence of primary amino acid. The active site amino acids are assembled to the cluster in the right conformation by the 3-dimensional folding of the primary amino acid sequence. The most frequent active site amino acid residues out of the 20 amino acids forming the protein are polar amino acids, aspartate, cysteine, glutamate, histidine, Serine, and lysine. Typically, only 2-3 essential amino acid residues are involved directly in the bond causing the formation of the product. Glutamate, Aspartate, and Histidine are the amino acid residues which also serve as a proton acceptor or donor.

Temperature and pH

Enzymes require an optimum temperature and pH for their action. The temperature or pH at which a compound shows its maximum activity is called optimum temperature or optimum pH, respectively. As mentioned earlier, enzymes are protein compounds. A temperature or pH more than optimum may alter the molecular structure of the enzymes. Generally, an optimum pH for enzymes is considered to be ranging between 5 and 7.

• The greatest number of molecular collisions
• human enzymes = 35°- 40°C
• body temp = 37°C
• Heat: increase beyond optimum T°
• The increased energy level of molecule disrupts bonds in enzyme & between enzyme & substrate H, ionic = weak bonds
• Denaturation = lose 3D shape (3° structure)
• Cold: decrease T°
• Molecules move slower decrease collisions between enzyme & substrate

Concentration and Type of Substrate

Enzymes have a saturation point, i.e., once all the enzymes added are occupied by the substrate molecules, its activity will be ceased.  When the reaction begins, the velocity of enzyme action keeps on increasing on further addition of substrate. However, at a saturation point where substrate molecules are more in number than the free enzyme, the velocity remains the same.

The type of substrate is another factor that affects the enzyme action. The chemicals that bind to the active site of the enzyme can inhibit the activity of the enzyme and such substrate is called an inhibitor. Competitive inhibitors are chemicals that compete with the specific substrate of the enzyme for the active site. They structurally resemble the specific substrate of the enzyme and bind to the enzyme and inhibit the enzymatic activity. This concept is used for treating bacterial infectious diseases.

Salt concentration

Changes in salinity: Adds or removes cations (+) & anions (–)

• Disrupts bonds, disrupts the 3D shape
• Disrupts attractions between charged amino acids
• Affect 2° & 3° structure
• Denatures protein
• Enzymes intolerant of extreme salinity
• The Dead Sea is called dead for a reason

Functions of Enzymes

The enzymes perform a number of functions in our bodies. These include:

• Enzymes help in signal transduction. The most common enzyme used in the process includes protein kinase that catalyzes the phosphorylation of proteins.
• They break down large molecules into smaller substances that can be easily absorbed by the body.
• They help in generating energy in the body. ATP synthase is the enzyme involved in the synthesis of energy.
• Enzymes are responsible for the movement of ions across the plasma membrane.
• Enzymes perform a number of biochemical reactions, including oxidation, reduction, hydrolysis, etc. to eliminate the non-nutritive substances from the body.
• They function to reorganize the internal structure of the cell to regulate cellular activities.

Frequently Asked Questions

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Mechanism of Enzyme Action

The mechanism of enzyme action depends upon the two factors, namely enzyme’s specificity and transition state of the reactants or substrates. The enzyme’s specificity is due to its active site, which seems like a small aperture or opening. The enzyme’s active site allows specific binding of an enzyme with the substrate due to residues like –NH 2 , -SH groups etc.

We must have heard that the enzymes are the biocatalysts, but we need to know what biocatalysts do. The participation of enzymes in any biochemical or biological reaction is referred to as “ Catalyzed reaction ”, and they only speed up the reaction upto 10 7 to 10 20 times.

Therefore, enzymes serve as a biocatalyst that only increases the reaction rate or the conversion of reactants into products. It is important to keep in mind that the enzymes are never used up in the reaction, which means they remain free after the release of products.

Enzymes can catalyze the same chemical pathway several times until they get denatured and associate with the inhibitors. In this context, we will study the mechanism of enzyme action through three popular models (lock and key hypothesis, Induced fit model and Michaelis and Menten’s equation.

You will also get to know the difference between the mechanisms of enzyme-catalyzed and uncatalyzed reaction (without enzyme) along with the meaning of some important terms relative to the study of the enzyme’s mechanism.

Content: Mechanism of Enzyme Action

Important terms, lock and key hypothesis, induced fit model, michaelis and menten’s model.

Before proceeding to the mechanism of enzyme action, we must have a brief knowledge of the following terms:

Enzymes : These are the 3-D proteinaceous organic compounds, which function as a “ biocatalyst ” to increase the reaction’s speed. Enzymes are specific due to the presence of a distinct region called an active site of an enzyme .

Enzyme action : It is defined as the enzyme’s activity, which facilitates the catalysis or breakdown of chemical substrates (participating in the reaction) into the desired products. Therefore, the term “Enzyme action” is sometimes interchangeable with the term “ Enzyme catalysis ”.

Enzyme catalysis is necessary for many biological or biochemical pathways to occur or essential for the chemical interconversions that sustain life. Let us look into a few examples of enzyme catalysis:

• Sucrose (disaccharide) converts into two different monosaccharide molecules, i.e. glucose and fructose, via the enzyme action of “ Sucrase ”.
• Glucose (monosaccharide) converts into ethanol (primary alcohol) and atmospheric carbon dioxide via the action of the enzyme “ Zymase ”.

Substrates : In terms of enzymology, the substrates refers to the reactants molecules, which form a temporary association with an enzyme or turn out to form an enzyme-substrate complex ( E-S complex ). Various bonds form between the initial contact of the two, i.e. an enzyme and substrate that releases binding energy to create a perfect fit.

Products : In terms of enzymology, the products refer to the species or molecules form by the conformation changes in the enzyme-substrate complex. The enzymes attain their original state after the release of products, and they are available for the substrate molecules to undergo the same pathway.

The mechanism of enzyme action typically depends upon the activation energy. Enzymes participating in any chemical reaction reduce the activation energy and decreasing the time between the substrate’s interconversion into a product. Therefore, to study the enzyme’s mechanism more in detail, we must know the meaning of the following terms:

Transition state : It refers to the high energy state during which the substrates are in the process of falling into the products. The transition state is the intermediary stage between the substrate and product, which remains unstable , or this stage does not last for long. The substrates require some activation energy to outreach the transition state.

Activation energy : It refers to the minimum energy required for the substrates to get into the transition state and contort the substrate molecules into the desired products. Reactants can form products by utilizing the heat energy from the surroundings. But, the reactants in association with enzymes release products more rapidly.

Catalysis : Any chemical reaction, which uses a biocatalyst or heat energy to contort the substrates into products, is the process called catalysis. Substrate transforming into the products solely by the heat energy comes under the category of the uncatalyzed reaction .

Oppositely, substrates contorting into products by the participation of a biocatalyst (enzyme) comes under the category of the catalyzed reaction . Enzymes lower the activation energy or increase the reaction rate (conversion of substrate into a product).

Free energy : In terms of enzymology, free energy or Gibbs free energy is the potential difference between substrates and products’ energy level. It is denoted as ∆G .

It was pioneered by a scientist named Emil Fischer (in 1894), which explains the enzyme’s mechanism. According to this model, an active site is a region of the enzyme, which bears a specific shape or conformation.

Lock and key hypothesis have a simple approach, which says that the particular substrate perfectly fits into the enzyme’s cleft ( active site ) for the reaction to occur. Similarly, the way one specific key fits into the notch of a lock and unlocks it.

The amino acid residues enable the enzyme’s active site to bind specifically with the substrate. Thus, this model explains an enzyme’s specificity to which only the substrate can bind those with a shape corresponding to an active site’s shape. The lock and key model has many loopholes like:

• This experiment fails to explain the broad specificity of an enzyme.
• It did not explain the binding mechanism of the substrate with an enzyme.
• The lock and key model could not give any information about the mechanism of enzyme catalysis or product formation.

It is the widely accepted model to study the mechanism of enzyme action and pioneered by the scientist Daniel Koshland (in 1959). According to his theory, an active site is a flexible region of the enzyme, which can undergo conformational changes. It is also popular by the name of the hand in glove model .

The induced-fit model explains that the enzyme’s active site possesses two specific locations (buttressing and catalytic site). The substrate initially attaches to the buttressing region , after which the catalytic site brings some conformational changes in the E-S complex. The conformation changes result in the breaking of various bonds between the two and cause the product’s formation.

After the catalysis, the enzyme becomes free to carry out the new cycle of converting the substrates into the products. Thus, the induced fit model compensates for the lock and key theory’s loopholes by explaining the broad specificity of an enzyme and the catalysis of the reaction.

Leonor Michaelis and Maud Menten gave an equation in 1913 to explain the mechanism of enzyme action. It depends upon the lowering of activation energy . According to Michaelis Menten’s equation, the enzyme-substrate complex can reversibly dissociate into (enzyme plus substrate) and further proceed to give (enzyme plus product).

In a catalyzed reaction or an enzyme’s presence, the substrate rapidly reaches the transition state due to decreased activation energy. The enzyme reduces the energy required (activation energy) for the substrate to form products. Conversely, the substrates take more time to reach the transition state and form products without an enzyme catalyst.

The Gibbs free energy will not change even by the participation of an enzyme. Therefore, the Gibbs energy for both catalyzed and uncatalyzed reactions will remain the same . Thus, this model also explains the speed of the reaction.

We can conclude that the mechanism of enzyme action is to lower the activation energy or speed up the substrate’s interconversion into a product. No chemical reaction does not utilize enzymes, unlike substrates, or the enzymes remain free to carry out more chemical interconversions.

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April 2, 2024

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'Unheard of in structural biology': New enzyme models reveal disease insights

by The Scripps Research Institute

When nucleic acids like DNA or RNA build up in a cell's cytoplasm, it sets off an alarm call for the immune system. Enzymes usually clear these nucleic acids before they cause an issue, but when these enzymes don't work and the immune system gets called in, it can lead to autoimmune and inflammatory diseases.

In a new study published in Structure , Scripps Research scientists present the previously undescribed structure of two of these nucleic acid-degrading enzymes—PLD3 and PLD4. Understanding these enzymes' structures and molecular details is an important step toward designing therapies for the various diseases that arise when they malfunction, which include lupus erythematosus, rheumatoid arthritis and Alzheimer's disease.

"These enzymes are important for cleaning up the cellular environment, and they also set the threshold for what is considered an infection or not," says senior author David Nemazee, Ph.D., professor in the Department of Immunology and Microbiology at Scripps Research. "I'm hoping someday we may be able to help patients based on this information."

Enzymes are proteins that speed up chemical reactions by binding and reacting to specific molecules called substrates. In the case of PLD3 and PLD4, the substrate is a strand of RNA or DNA, which the enzymes break down nucleotide by nucleotide.

The team used X-ray crystallography to build atomic-scale models of the PLD3 and PLD4 in multiple states or situations, allowing them to examine how their shapes changed over the course of the catalytic reaction. This included when the enzymes were resting, or when they were actively bound to a substrate.

"These models allow us to visualize PLD3 and PLD4 very clearly and with high resolution, so we know exactly how every atom interacts, meaning we can deduce how the enzymes work," says first author Meng Yuan, a staff scientist in the Department of Integrative Structural and Computational Biology at Scripps Research.

The structural analyses revealed that PLD3 and PLD4 are structurally similar and that they degrade DNA and RNA in a very similar fashion, even though PLD4 is a larger protein. Both enzymes degrade nucleic acids via a two-step process.

"We call this process a two-step catalysis: bite down and release," says Yuan. "In the first step, the enzyme bites down on the DNA strand and separates a single 'brick' or nucleotide from the rest of the strand, and in the second step, it opens its 'mouth' and releases the brick to be recycled."

Because the enzymatic reaction happens so quickly—within milliseconds—researchers needed to use an alternative substrate to visualize the enzymes' structure during catalysis. To do this, they incubated the enzymes together with a molecule that looks very similar to the DNA that the enzyme usually degrades, but that the enzymes degrade much more slowly.

This method uncovered a previously unknown function for one of the enzymes: In addition to biting off nucleotides from single-stranded RNA and DNA, PLD4 also showed phosphatase activity, which means it might also be involved in breaking down DNA's phosphate backbone.

"I think it's amazing that the crystal structure told us about this phosphatase activity," says Nemazee. "To discover new enzymatic activity is unheard of in structural biology. It's only because Meng was able to solve such an amazingly accurate and detailed structure that he could inform us about this extra enzymatic activity that we had no idea about."

After they had elucidated PLD3 and PLD4's usual structure, the researchers examined the structure of variants that are associated with diseases, including Alzheimer's and spinocerebellar ataxia. These analyses revealed that some of these variants had decreased enzymatic capability, while others—including a mutation associated with late-onset Alzheimer's—appeared to be more active.

"Some of our data suggests that one of these Alzheimer's-associated enzyme variants might function better, which was a surprise to me, but it also may be less stable and more easily aggregated," says Nemazee.

The researchers plan to continue investigating the structure and function of these enzymes. Their next steps include exploring possible ways of inhibiting the enzymes in scenarios where they are overactive, and they also plan to investigate the possibility of replacing the enzymes in people who carry non-functional (or non-working) versions.

In addition to Nemazee and Yuan, authors of the study, "Structural and mechanistic insights into disease-associated endolysosomal exonucleases PLD3 and PLD4," were Linghang Peng, Deli Huang, Amanda Gavin, Fangkun Luan, Jenny Tran, Ziqi Feng, Xueyong Zhu, Jeanne Matteson, and Ian Wilson, all of Scripps Research.

Journal information: Structure

Provided by The Scripps Research Institute

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