Enzyme Specificity

11 Key excerpts on "Enzyme Specificity"

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  eBook - PDF

  Biochemistry and its application

  • Papita H Gourkhede(Author)
  • 2023(Publication Date)
  • Arcler Press

    (Publisher)

  Biochemistry and its Application 18 1.3.2. Properties of Enzymes Active site: Enzyme molecules contain a special pocket or cleft called the active site. The active site contains amino acid chains that create a three- dimensional surface complementary to the substrate. The active site binds the substrate, forming an enzyme-substrate (ES) complex. ES is converted to enzyme-product (EP); which subsequently dissociates to enzyme and product. For the combination with the substrate, each enzyme is said to possess one or more active sites where the substrate can be taken up. The active site of the enzyme may contain a free hydroxyl group of serine, phenolic (hydroxyl) group of tyrosine, SH-thiol (Sulfhydryl) group of cysteine, or imidazole group of histidine to interact with there is substrates. It is also possible that the active site (Catalytic site) is different from the binding site in which case they are situated closely together in the enzyme molecule. Catalytic efficiency/ Enzyme turnover number: Most enzyme-catalyzed reactions are highly efficient proceeding from 103 to 108 times faster than uncatalyzed reactions. Typically, each enzyme molecule is capable of transforming 100 to 1000 substrate molecules into products each second. Enzyme turnover number refers to the amount of substrate converted per unit time (carbonic anhydrase is the fastest enzyme). Specificity: Each enzyme has a specific substrate. The specificity of enzymes is divided into a. Absolute specificity:- this means one enzyme catalyzes or acts on only one substrate. For example, Urease catalyzes the hydrolysis of urea but not thiourea. b. Stereo specificity- some enzymes catalyze only one type of molecule, even if the compound is one type of molecule: glucose oxidase catalyzes the oxidation of β-D-glucose but not α-D-glucose, and arginase catalyzes the hydrolysis of L-arginine but not D-arginine. Maltase catalyzes the hydrolysis of α- but not β –glycosides.

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  Enzymes

  A Practical Introduction to Structure, Mechanism, and Data Analysis

  • Robert A. Copeland(Author)
  • 2004(Publication Date)
  • Wiley-Interscience

    (Publisher)

  L. (1913) Biochem. Z. 49 333. Schulz, A. R. (1994) Enzyme Kinetics from Diastase to Multi-enzyme Systems, Cambridge University Press, New York. Segel, I. H. (1975) Enzyme Kinetics, Wiley, New York. Wahl, R. C. (1994) Anal. Biochem. 219, 383. Wilkinson, A. J., Fersht, A. R., Blow, D. M., and Winter, G. (1983) Biochemistry, 22, 3581. REFERENCES AND FURTHER READING 145 6 CHEMICAL MECHANISMS IN ENZYME CATALYSIS The essential role of enzymes in almost all physiological processes stems from two key features of enzymatic catalysis: (1) enzymes greatly accelerate the rates of chemical reactions; and (2) enzymes act on specific molecules, referred to as substrates, to produce specific reaction products. Together these properties of rate acceleration and substrate specificity afford enzymes the ability to perform the chemical conversions of metabolism with the efficiency and fidelity required for life. In this chapter we shall see that both substrate specificity and rate acceleration result from the precise three-dimensional structure of the substrate binding pocket within the enzyme molecule, known as the active site. Enzymes are (almost always) proteins, hence the chemically reactive groups that act upon the substrate are derived mainly from the natural amino acids. The identity and arrangement of these amino acids within the enzyme active site define the active site topology with respect to stereochemis- try, hydrophobicity, and electrostatic character. Together these properties define what molecules may bind in the active site and undergo catalysis. The active site structure has evolved to bind the substrate molecule in such a way as to induce strains and perturbations that convert the substrate to its transition state structure. This transition state is greatly stabilized when bound to the enzyme; its stability under normal solution conditions is much less.

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  Introduction to Modern Biochemistry 4e

  • P Karlson(Author)
  • 2012(Publication Date)
  • Academic Press

    (Publisher)

  The last two enzymes happen to use the same coenzyme (pyridoxal phosphate, Chapter VIII,4). The reaction specificity therefore depends on the protein com-ponent, not on the coenzyme (cf. Chapter VI, 1). This selectivity of reactions by the enzymes is of prime importance for life processes in general. 6. SPECIFICITY OF ENZYME CATALYSIS 85 The following mechanism furnishes a likely (but still incomplete) explanation of reaction specificity: Enzyme and substrate form a weak bond from which the reaction on the substrate may then proceed. The kind of bond and/or steric arrangement between enzyme and substrate prepares for the specific reaction. In bimolecular reactions, two substrates must be bound simultaneously to the enzyme surface to allow the substrates to react with each other. Frequently, one of the reaction partners is a coenzyme (see Chapter VI). Substrate Specificity. The principle of selection operates furthermore in the binding of the substrate to the enzyme. Certainly not every substance which might be able to undergo a particular reaction is bound. In the example of the amino acid decarboxylase, a few amino acids are bound tightly, some loosely, and others not at all. The latter do not react, i.e., their decarboxylation is not catalyzed. This kind of selectivity is called substrate specificity. It is especially characteristic for optical antipodes (mirror-image isomers). Usually only one of the two isomers reacts, or at least one antipode reacts much faster. The extent of substrate specificity varies from enzyme to enzyme. A few hydrolases are relatively nonspecific, others require substrates containing certain groups (group specificity; e.g., jS-galactosidase and α-glucosidase, which cleave all β-galactosides and α-glucosides, respectively; cf., Chapter XVII,3).

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  Protein Engineering Handbook

  • Stefan Lutz, Uwe Theo Bornscheuer(Authors)
  • 2012(Publication Date)
  • Wiley-VCH

    (Publisher)

  5 -fold, Koshland proposed a more dynamic view of enzyme action. This revised picture of selectivity became known as the ‘induced fit’ theory [5]. According to this model, the substrate itself plays an active role in discrimination by inducing changes in the tertiary structure of the catalyst, which act to align catalytic groups in a configuration that is conducive to catalysis. The validity of induced fit as a structural model for Enzyme Specificity was borne out when the first crystal structures of yeast hexokinase came to light during the 1970s [6, 7]. Since that time, a great number of enzymes have been shown to undergo measurable conformational changes during substrate association. In many cases, these conformational changes function to envelop the substrate completely, so as to remove any interactions with bulk solvent water.

  Despite its apparent widespread acceptance in biochemistry circles, several investigators have suggested that induced fit conformational changes fail to provide a satisfactory thermodynamic explanation for Enzyme Specificity [8–10]. These arguments are based largely upon the assumption that the transition states through which competing substrates proceed are chemical in nature and involve the acquisition of identical enzyme conformations. The extent to which enzymatic transformations generally adhere to these requirements is unknown. As was noted in Section 13.1, it seems unlikely that the same microscopic step universally limits the transformation rates of both good and poor substrates. Nevertheless, the debate regarding the validity of the induced fit concept is ongoing in the literature, and the development of a consistent structural, kinetic and thermodynamic model for Enzyme Specificity remains an active area of biochemical research [2, 11–13].

  13.3 Advantages and Disadvantages of Specificity

  The advantages of Enzyme Specificity in biological systems are numerous. By providing a wealth of potential sites of regulation within individual pathways, the specialization of catalytic function has enabled the attainment of an unprecedented level of control over metabolic flux in living systems. In addition, enzyme specialization has allowed single enzymes the opportunity to achieve high levels of catalytic efficiency, thereby maximizing turnover rates. The specialization of function, however, comes at a cost. To synthesize a multitude of polypeptides, each specialized for a single purpose, is metabolically expensive both at the level of translation and regulation. In principle, high levels of specialization may also impede the rapid evolution of new function. However, contemporary processes such as the acquisition of antibiotic resistance by pathogenic bacteria and the bioremediation of anthropogenic toxins by microbes suggest that certain organisms retain the ability to rapidly adapt to new metabolic challenges, despite the presumed specialization of their constituent protein catalysts.

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  Introduction to Proteins

  Structure, Function, and Motion, Second Edition

  • Amit Kessel, Nir Ben-Tal(Authors)
  • 2018(Publication Date)
  • Chapman and Hall/CRC

    (Publisher)

  catalysis ). As we will see below, they do so by promoting the conversion of the substrate either into its transition state or into another intermediate downstream of the reaction coordinate. This is done either through chemical reactions between substrate and active site groups (amino acids, cofactors) or via noncovalent stabilization of reaction intermediates by these groups. The latter shows that binding and catalysis are often not mutually exclusive.

  Substrate binding and catalysis often take place in a single location inside the active site. However, when the substrate is large or has a complex structure, the enzyme may contain multiple binding sites, each binding a different part of the substrate. Still, only one of these sites carries out the catalysis, specifically, the site that binds the labile part of substrate that is supposed to be chemically transformed.

  As explained above, both substrate binding and catalysis are carried out by chemical groups in the enzyme’s active site, which either react or interact specifically with the substrate. This specificity is created by the shape of the active site and the spatial distribution of its chemical groups. Indeed, enzymatic active sites have been evolutionarily selected to complement their natural substrates

  [150 ]

  , both geometrically and electrostatically

  [151 –153 ]

  *1

  . This complementarity is responsible for the selectivity of enzymes towards their cognate substrates over other molecules, as well as for their catalytic efficiency and reaction specificity. These key aspects are further discussed in the subsections below.

  9.3.2 Binding specificity and selectivity

  Enzymes are known to bind their substrates selectively, i.e., to favor cognate substrates over non-cognate ones. Selectivity exists both in the ground state (which we discuss here) and in the transition state (which we discuss in Subsection 9.3.3.2 below) of the substrate, with selectivity in the transition state being stronger

  [153 ]

  . In both cases, the selectivity results from attractive and repulsive

  *2

  noncovalent interactions between the active site and substrate, with attractive interactions being stronger with cognate substrates, and repulsive interactions, which have a larger effect, being stronger with non-cognate substrates [

  153

  ]

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  Cell Chemistry and Physiology: Part I

  • Edward Bittar(Author)
  • 1996(Publication Date)
  • Elsevier Science

    (Publisher)

  How Enzymes Work 31 fish (vertically mounted). Consider how many changes you would have to make in your left hand to accommodate a D-amino acid by the interactions outlined above. Thus, once biological specificity has been set, it is very difficult to change. These very cursory examples illustrate the phenomenon of binding of molecules to others in a biological system. Although these types of binding events are described for enzymes--the topic of this chapter, there are many other binding events that do not result in a chemical reaction. However, all binding events are based on several points of contact between one molecule (often a protein) and another. In every case, the binding process produces specificity for the molecule that is bound. This makes specificity one of the foundation properties of biology. Specificity generated by binding is used for many purposes, including recogni- tion of one's own molecules and rejection of foreign molecules. Such recognition can be illustrated by the immune system which binds foreign materials and targets them for destruction. A high profile example is the rejection of tissue transplants and the need for tissue donors with compatible structures. However, biological organisms at all levels, including bacteria, display specificity. For example, many ~rganisms produce DNA molecules with special properties or derivatives so they can recognize foreign DNA and degrade it. Thus, specificity and rejection of foreign material is common throughout biology. Overall, while specificity has many purposes, it probably arose first because it is an unavoidable outcome of biological catalysis. You cannot have enzyme catalysis without binding of substrates and you cannot create a binding site without creating specificity.

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  Biomolecules

  From Genes to Proteins

  • Shikha Kaushik, Anju Singh(Authors)
  • 2023(Publication Date)
  • De Gruyter

    (Publisher)

  Enzyme Specificity is essential for the normal functioning of cells as it regulates the metabolic pathways and prevents the unwanted side reactions at a particular active site. Enzymes showing highest specificity and accuracy also exhibit “proofreading” mechanisms and are involved in important biological processes like replication and gene expression. For example, DNA polymerase performs various crucial functions: it catalyzes the synthesis of DNA, checks the product, and also repairs it, if found any incorrect base pair.

  3.3  Mechanism of Enzyme Action

  In an enzyme-catalyzed reaction, the enzyme [E] binds to the substrate [S] to form a complex. The enzyme–substrate [ES] complex then forms the product [P]:

  E + S ⇌

  E S

  ⇌ P + E

  A substrate usually binds to the small region of the enzyme called active site. The active site is the region within the enzyme where the substrate molecule binds, undergoes chemical reaction, and releases products. It is usually a groove or cleft in the enzyme formed by the folding pattern of the protein. As most of the enzymes are proteins, and proteins are made up of amino acids, the amino acid side chains align themselves in a manner that they can bind to the substrate via noncovalent interactions such as hydrogen bonds, ionic interactions , hydrophobic interactions, and van der Waals interactions. The reactive side chains of aspartate, glutamate, cysteine, lysine, arginine, serine, threonine, histidine, and hydrophobic residues play a crucial role in substrate binding. The substrate bound to the enzyme undergoes a conformational change, attaining a temporary tense state from which it transformed into products. Although the active site is a small portion of the enzyme involving only few amino acids in the catalysis process, it is the complete three-dimensional structure of protein which contributes to maintain a proper configuration at the catalytic site. The catalyzed reaction occurs at the active site of an enzyme in various steps as illustrated in Figure 3.1 :

  1. The first step involves the binding of a substrate to the active site of the enzyme.

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  Enzyme Inhibition and Bioapplications

  • Rakesh R. Sharma(Author)
  • 2012(Publication Date)
  • IntechOpen

    (Publisher)

  Conceptually, enzyme inhibitors are classified into two types: non-specific inhibitors and specific inhibitors. The enzyme inhibition reactions follow a set of rules as mentioned in following rules. Presently, computer based enzyme kinetics data analysis softwares are developed using following basic presumptions. 1. Enzyme interacts with substrate in 1:1 ratio at active site to catalyze the reaction. 2. Enzyme binds with substrate at active site in the form of a lock-key 3D arrangement for induced fit. 3. Inhibitor active groups compete with substrate active groups and/or active groups at enzyme allosteric catalytic site in a synergistic manner or first cum first preference (competition) to make enzyme-inhibitor-substrate/enzyme-substrate/enzyme-inhibitor complexes. 4. Enzyme-inhibitor-substrate complex formation depends on active free energy loss and thermodynamic principles. 5. Enzyme and substrate or inhibitors react with each other as active masses and reaction progresses in kinetic manner of forward or backward reaction. 6. Kinetic nature of inhibitor or substrate binding with enzyme is expressed as kinetic constants of a catalytic reaction. 7. Enzyme reaction(s) are highly depend on physiological conditions such as pH, temperature, concentration of reactants, reaction period to determine the rate of reaction. 8. Substrate and inhibitor molecules arrange over enzyme active site on specific sub unit(s) in 3D manner. As a result enzyme-substrate-inhibitor exhibit binding rates depend on allosteric sites or subunit-subunit homotropic or heterotropic interactions. 9. Intermolecular forces between enzyme subunits, substrate or inhibitor active group interactions, physical properties of binding nature: electrophilic, hydrophilic, nucleophilic and metalloprotein nature; hydrogen bonding affect the overall enzyme reaction rates and mode of inhibition (3D orientation of inhibitor molecule on enzyme active site).

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  Molecular Aspects of Enzyme Catalysis

  • Toshio Fukui, Kenji Soda, Toshio Fukui, Kenji Soda(Authors)
  • 2008(Publication Date)
  • Wiley-Blackwell

    (Publisher)

  New Trends in Enzyme Studies 11 As a whole, such high stereospecificity is difficult to attain by chemical catalysis, although asymmetric synthesis has developed markedly in recent years.) Stereospecificity has been applied to the production of optically active compounds such as amino acids, and also to the synthesis of stereospecifically isotope-labeled compounds, the synthesis of which is not easily achieved by a chemical method (Table I. 3).75) Enzymes play a central role in biotechnological processes in the production of useful materials other than those presented above, and enzyme sensors in which enzyme reaction is coupled with an electrochemical monitoring system are used clinical, process and environmental analyses. It is, however, out of the scope of this section to review these topics and other reviews should be consulted.8') Perspectives The studies were laborious and time-consuming. This situation has been greatly improved by innovations such as the HPLC system and microcapillary electrophoresis, which have enabled the purification of a small amount of enzyme effectively. We in general can isolate and purify most enzymes by a combination of gene technology with these methods without difficulty. Increase in sensitivity and efficiency of analysis in structural studies of enzymes with a gas phase sequencer have made it possible to determine the primary structure in a shorter period of time with a small amount of enzyme at the picomole or even femtomole level. In addition, thanks to the DNA sequencing technique the number of enzymes (or proteins) whose amino acid sequences are registered in a data base file has expanded explosively. The introduction of mass spectrometry on the primary structure determination of protein has stimulated the search for a new methodology other than Edman chemistry.

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  Biochemical Engineering

  • Douglas S. Clark, Harvey W. Blanch(Authors)
  • 1997(Publication Date)
  • CRC Press

    (Publisher)

  Chapter 1. Enzyme Catalysis Enzymes are one of the essential components of all living systems. These macromo- lecules have a key role in catalyzing the chemical transformations that occur in all cell metabolism. The nature and specificity of their catalytic activity is primarily due to the three-dimensional structure of the folded protein, which is determined by the sequence of the amino acids that make up the enzyme. The activity of globular proteins may be regulated by one or more small molecules, which cause small conformational changes in the protein structure. Catalytic activity may depend on the action of these non-protein components (known as cofactors) associated with the protein. If the cofactor is an organic molecule, it is referred to as a coenzyme. The catalytically inactive enzyme (without cofactor) is termed an apoenzyme; when coenzyme or metal ion is added, the active enzyme is then termed a holoenzyme. Many cofactors are tightly bound to the enzyme and cannot be easily removed; they are then referred to as prosthetic groups. In this chapter we shall examine the nature of enzyme catalysis, first by examining the types of reactions catalyzed and the mechanisms employed by enzymes to effect this catalysis, and then by reviewing the common constitutive rate expressions which describe the kinetics of enzyme action. As we shall see, these can range from simple rate expressions to complex expressions that involve several reactants and account for modification of the enzyme structure. 1.1 Specificity of Enzyme Catalysis Enzymes have been classified into six main types, depending on the nature of the reaction catalyzed. A numbering scheme for enzymes has been developed, in which the main classes are distinguished by the first of four digits. The second and third digits describe the type of reaction catalyzed, and the fourth digit is employed to distinguish between enzymes of the same function on the basis of the actual substrate in the reaction catalyzed.

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  Evaluation of Enzyme Inhibitors in Drug Discovery

  A Guide for Medicinal Chemists and Pharmacologists

  • Robert A. Copeland(Author)
  • 2013(Publication Date)
  • Wiley-Interscience

    (Publisher)

  2. The active site is three-dimensional—that is, amino acids and cofactors in the active site are held in a precise arrangement with respect to one another and with respect to the structure of the substrate molecule. This active site three-dimensional structure is formed as a result of the overall tertiary structure of the protein.

  3. In most cases the initial interactions between the enzyme and the substrate molecule (i.e., the initial binding event) are noncovalent, making use of hydrogen bonding, electrostatic, hydrophobic interactions, and van der Waals forces to effect binding.

  4. The active site of enzymes usually are located in clefts and crevices in the protein. This design effectively excludes bulk solvent (water), which would otherwise reduce the catalytic activity of the enzyme. In other words, the substrate molecule is desolvated upon binding, and shielded from bulk solvent in the enzyme active site. Solvation by water is replaced by specific interactions with the protein (Warshel et al., 1989).

  5. The specificity of substrate utilization depends on the well-defined arrangement of atoms in the enzyme active site that in some way complements the structure of the substrate molecule.

  These features of enzyme active sites have evolved to facilitate catalysis by (1) binding substrate molecules through reversible, noncovalent interactions, (2) shielding substrate molecules from bulk solvent and creating a localized dielectric environment that helps reduce the activation barrier to reaction, and (3) binding substrate(s) in a specific orientation that aligns molecular orbitals on the substrate molecule(s) and reactive groups within the enzyme active site for optimal bond distortion as required for the chemical transformations of catalysis (see Copeland, 2000, for a more detailed discussion of these points). These same characteristics of enzyme active sites make them ideally suited for high-affinity interactions with molecules containing the druggable features described earlier (Taira and Benkovic, 1988).

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