Enzyme Activity

11 Key excerpts on "Enzyme Activity"

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  Medical Biochemistry

  • Gustavo Blanco, Antonio Blanco(Authors)
  • 2017(Publication Date)
  • Academic Press

    (Publisher)

  transition intermediary , which will be subjected to catalysis.

  Zymogens or proenzymes are inactive precursors of enzymes. They acquire activity after hydrolysis of a portion of their molecule.

  Cellular location of enzymes varies, the majority being in different compartments of the cell, while others are extracellular.

  Multienzyme systems are those composed of a series of enzymes or enzyme complexes. There are also multifunctional enzymes with several different catalytic sites in the same molecule.

  Enzyme Activity is determined by measuring the amount of product formed, or substrate consumed in a reaction in a given time. Initial velocity corresponds to the activity measured when the amount of consumed substrate is less than 20% of the total substrate originally present. One IU of enzyme catalyzes the conversion of 1 μmol of substrate per second under defined conditions of pH and temperature. Specific activity is the units of enzyme per milligram of protein present in the sample. Molar activity or turnover number are the substrate molecules converted into product per unit time per enzyme molecule, under conditions of substrate saturation.

  The rate of the enzymatic reaction is directly proportional to the amount of enzyme rate present in the sample.

  Also, at low [S] and under constant conditions of the medium, Enzyme Activity rapidly increases with the raise in [S]. At higher substrate levels, the activity increases slowly and tends to reach a maximum. The effect follows a hyperbolic

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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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  Molecular Biophysics

  • M Volkenstein(Author)
  • 2012(Publication Date)
  • Academic Press

    (Publisher)

  Molecular activity can also be expressed as the turnover rate (Warburg), which is the number of moles of substrate transformed per mole of enzyme per minute. Methods for extracting and purifying enzymes are well ad-vanced, and many enzymes have been obtained in the pure crystal-line form [15,30,31]. Thus it is possible to study reactions and structures of enzymes in vitro. The situation in vitro dif-fers from that in the cell because the cell is an open system and à great number of enzymic reactions, including coupled re-actions, proceed there simultaneously. However, the study of enzymes in vitro provides a strong foundation for understanding the corresponding biological processes. When an ESC (enzyme-substrate complex) is formed, the small substrate molecule (or molecules) is (are) bound stoichiometri-cally to a large enzyme molecule. Evidently the substrate binds directly to some specific small region of the enzyme molecule, called the active site. The nature of the active site, that is, the nature of the amino acid residues and their positions, as well as of the cofactors, participating at the active site, can be established by chemical and physical methods. The altera-tions in activity produced by chemical modifications of a pro-tein make it possible to determine the functional groups of the active site. Information about its structure can be obtained by means of spectrophotometry, spectropolarimetry, NMR and EPR spectroscopy (in the latter a paramagnetic label is introduced), etc. X-ray structural analysis reveals the actual geometric pattern of enzyme-substrate interaction. The diversity of amino acid residues and atomic groups of cofactors determines the polyfunctional properties of the active site, its ability to bind molecules of the substrate or modifier, and the catalytic activity [31].

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  Chemistry for Today

  General, Organic, and Biochemistry

  • Spencer Seager, Michael Slabaugh, Maren Hansen, , Spencer Seager, Spencer Seager, Michael Slabaugh, Maren Hansen(Authors)
  • 2021(Publication Date)
  • Cengage Learning EMEA

    (Publisher)

  For example, an enzyme preparation with an activity corresponding to 40 IU contains a concentration of enzyme 40 times greater than the standard. This is a useful way to mea- sure Enzyme Activity because the level of Enzyme Activity compared with normal activity is significant in the diagnosis of many diseases (see Section 20.9). Example 20.2 Describing Enzyme Activity List two ways of describing Enzyme Activity. Solution Turnover number and enzyme international unit. ✔ LEARNING CHECK 20.2 Differentiate between the terms turnover number and enzyme international unit. 20.6 Factors Affecting Enzyme Activity Learning Objective 6 Identify the factors that affect Enzyme Activity. Several factors affect the rate of enzyme-catalyzed reactions (see Figure 20.7). The most important factors are enzyme concentration, substrate concentration, temperature, and pH. In this section, we’ll look at each of these factors in some detail. In Section 20.7, we’ll consider another very important factor, the presence of enzyme inhibitors. 20.6A Enzyme Concentration In an enzyme-catalyzed reaction, the concentration of enzyme is normally very low com- pared with the concentration of substrate. When the enzyme concentration is increased, the concentration of ES also increases in compliance with reaction rate theory: E 1 S S d ES increased [E] gives more [ES] Thus, the availability of more enzyme molecules to catalyze a reaction leads to the for- mation of more ES and a higher reaction rate.

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  Cell Chemistry and Physiology: Part I

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

    (Publisher)

  Chapter 2 How Enzymes Work GARY L. N ELSESTU EN Introduction 25 Free Energy of a Chemical Reaction 26 Step I of Enzyme Catalysis 28 Enzymes Bind Substrates and Align Them for Chemical Reaction 28 Catalysis by an Enzyme Active Site Produces the Phenomenon of 'Saturation Kinetics' 31 Use of Biological Specificity to Create Pharmaceuticals and Other Bioactive Molecules 34 Step 2 of Enzyme Catalysis: Binding is Followed by Chemical Reaction 40 Summary 44 INTRODUCTION Enzymes are responsible for bringing about chemical reactions under the mild conditions that are essential to our existence. In the chemistry lab we often catalyze reactions with heat (refluxing or boiling water temperatures) and acid or base (often 0.1-6.0 molar H § or OH-. However, these conditions are not compatible with life as we know it. While some unusual organisms do exist at high temperatures and in slightly acidic or basic medium, they are the exceptions. Furthermore, even these organisms have adapted by creating ways of keeping the pH inside the cell close to neutral (1 x 10-7M H+). Most organisms, including humans, live at low tempera- Principles of Medical Biology, Volume 4 Cell Chemistry and Physiology: Part I, pages 25-44 Copyright 9 1995 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-805-6 25 26 GARY L. NELSESTUEN ture (< 37 ~ C) and close to neutral pH. Fluctuations of body temperature by only a few degrees or of pH by a few tenths of a pH unit can create serious conditions for humans. Thus, living organisms require catalysts that can bring about chemical reactions under these mild conditions. In turn, the ways that these catalysts function are responsible for many of the most fundamental properties of living organisms. These properties also help explain the action of many medicines and drugs (as well as herbicides, pesticides, poisons, and all types of bioreactive molecules), and the phenomenon of 'biological specificity'.

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  Laboratory Techniques with Reagents and Solutions

  • Chavan, U D(Authors)
  • 2021(Publication Date)
  • Daya Publishing House

    (Publisher)

  Specific activity is a measure of enzyme processivity, at a specific (usually saturating) substrate concentration, and is usually constant for a pure enzyme. For elimination of errors arising from differences in cultivation batches and/or misfolded enzyme etc. an active site titration needs to be done. This is a measure of the amount of active enzyme, calculated by e.g. titrating the amount of active sites present by employing an irreversible inhibitor. The specific activity should then be expressed as μmol min −1 mg −1 active enzyme. If the molecular weight of the enzyme is known, the turnover number , or μmol product sec −1 μmol −1 of active enzyme, can be calculated from the specific activity. The turnover number can be visualized as the number of times each enzyme molecule carries out its catalytic cycle per second. Related Terminology The rate of a reaction is the concentration of substrate disappearing (or product produced) per unit time (mol L −1 s −1 ). The % purity is 100% × (specific activity of enzyme sample / specific activity of pure enzyme). The impure sample has lower specific activity because some of This ebook is exclusively for this university only. Cannot be resold/distributed. Enzyme Assay and Activity 333 the mass is not actually enzyme. If the specific activity of 100% pure enzyme is known, then an impure sample will have a lower specific activity, allowing purity to be calculated. Types of Assay All enzyme assays measure either the consumption of substrate or production of product over time. A large number of different methods of measuring the concentrations of substrates and products exist and many enzymes can be assayed in several different ways. Biochemists usually study enzyme-catalysed reactions using four types of experiments: • Initial rate experiments : When an enzyme is mixed with a large excess of the substrate, the enzyme-substrate intermediate builds up in a fast initial transient.

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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)

  Michaelis-Menten formalism . For more in-depth discussions of enzyme kinetics, we direct the reader to more advanced books or articles on this topic.

  9.2.1 Basic concepts

  One of the simplest means of measuring an enzyme-catalyzed reaction is to track the change in substrate or product concentration over time, in order to obtain the rate, or velocity (), of the enzyme. This simple experiment yields the dependency shown in Figure 9.18.

  The rate of the enzyme-catalyzed reaction can be calculated from the plot as:

  9.4

  rate =

  d [ P ]

  dt

  (where d [P ] is the change in molar concentration of the product, and dt is the period of time over which this change takes place).

  The plot shows that the rate of the enzyme-catalyzed reaction diminishes with time. This may result from various factors, such as the decrease in substrate concentration, inhibition by the accumulating product, a change in pH , or thermal inactivation (reversible or irreversible (denaturation)

  [126 ]

  ). In any case, to be able to follow enzymatic activity, it is customary to measure the initial velocity (rate ) of the enzyme (V 0), i.e., the rate obtained at the beginning of the reaction, before it is influenced by external factors. V 0 depends on the concentrations of the enzyme and the substrate. When V 0

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  Bioanalytics

  Analytical Methods and Concepts in Biochemistry and Molecular Biology

  • Friedrich Lottspeich, Joachim W. Engels(Authors)
  • 2018(Publication Date)
  • Wiley

    (Publisher)

  The bonding of the enzyme to a conformation of the substrate that is analogous to the transition state and the resultant reduction in activation energy are obviously coupled to the tertiary and quaternary structure of the enzyme protein. As with all proteins, these are greatly dependent, in turn, on the environment, in particular on the pH and the ionic strength. The dependence of the enzymatic activity as compared to other reactions is striking here, while the temperature dependence of many enzymatic reactions, for instance, is similar to that of normal chemical reactions. To describe an enzymatic activity in a meaningful manner, the experimental conditions must therefore be described in detail.

  A further characteristic of the kinetics of enzyme-controlled reactions is the dependence on the substrate concentration. While, in the case of normal chemical reactions, the reaction rate can be increased largely arbitrarily by increasing the concentration of the parent substances, this does not apply to enzymatic reactions. In this case, when the reaction rate is plotted against the substrate concentration, the typical result is a hyperbolic curve that asymptotically approaches a limit value, the so-called maximum rate.

  3.6 Michaelis–Menten Theory

  In an attempt to explain the hyperbolic dependence of the initial rate of an enzymatic reaction on substrate concentration, Brown and Henry postulated in 1905 that an enzyme–substrate complex is formed in an initial reaction step, which then decomposes in a second step to form the product and the free enzyme. The influence of the back reaction, which is also catalyzed by the enzyme, can be disregarded, since the product concentration is still approximately zero at the beginning of the reaction:

  (3.10)

  Michaelis and Menten derived a theoretical interpretation of such a process on the basis of two essential assumptions. According thereto, a constant concentration of the enzyme–substrate complex is established in a thermodynamic equilibrium with enzyme and substrate, which is not disrupted by the further reaction to form the product. They further postulated that the reaction of the enzyme–substrate complex to form the product is much slower than the decomposition back to free enzyme and substrate. Secondly, the concentration of free substrate is constant and equal to the total substrate concentration because the substrate concentration is substantially greater than the enzyme concentration.

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  Practice and Theory of Enzyme Immunoassays

  • P. Tijssen(Author)
  • 1985(Publication Date)
  • Elsevier Science

    (Publisher)

  The differ- ence in the energy content in the transition and substrate state is called the Gibbs free energy of activation. If it is high, only a few molecules will have a momentous energy content sufficiently high to pass this activation barrier. A rise in temperature can increase the proportion of substrate molecules with enough energy to pass this barrier. The increase in the reaction constant is proportional to the collision rate and can be expressed with the Arrhenius formula: where k is the reaction rate (proportionality factor), Z the collision frequency, A a constant and T the absolute temperature. By multiply- ing A with the gas constant, R, the activation energy present per mole is obtained which is designated as a. The term e-IRrrepresents the fraction of molecules having sufficient energy for product forma- tion. Enzymes combine with their specific substrate in such a way that the activation energy a is decreased to a lower value of aI. For example, for the decomposition of H20z without catalysis, the activa- tion energy is 70 kJ/mol, whereas with the catalase (an enzyme with a very high turnover number) this becomes 7 kJ/mol. Since R = 8.314 J/mol.K, from eq. (1) it follows that the acceleration by the enzyme is: Some enzymes have group specificity, i.e. they act on different but closely related substrates, whereas others have absolute specificity. Though the active site occupies a relatively small portion of the complete enzyme, the other parts of the enzyme may have significant influences on the activity. This phenomenon has a direct bearing Ch. 9 Enzyme Activity I53 on EIA where regions outside the catalytic site are modified by or for the conjugation. Details of enzyme kinetics can be found in the review by Cornish-Bowden ( I 979).

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  Cell Biology A Comprehensive Treatise V4

  Gene Expression: Translation and the Behavior of Proteins

  • David M. Prescott(Author)
  • 2012(Publication Date)
  • Academic Press

    (Publisher)

  6 Principles of the Regulation of Enzyme Activity Peter J. Roach I. Introduction 203 II. Some Basic Concepts of Biochemical Regulation 205 A. Metabolic Regimes and Metabolic Control 205 B. Classification of Enzymic Reactions 207 C. Signals, Sensitivity, and Time Scales 211 III. Mechanisms of Controlling Enzyme Activity 218 A. Noncovalent Mechanisms 218 B. Covalent Modification of Enzymes 239 C. Regulation of Enzyme Concentration 258 IV. Integration of Metabolic Controls 260 A. Classifications of Metabolites 260 B. Patterns of Metabolic Regulation 269 V. Conclusion 284 References 285 I. INTRODUCTION Analysis of the functional significance of biological structures has long been an important aspect of biology. The last half-century, in fact, has provided so many advances in our knowledge of the structure of or-ganisms, their cells, and cellular components that we must now assess the physical and chemical properties of individual molecular species in rela-tion to physiological function. This chapter, then, will deal with the in-terpretation of the chemical properties of enzymes within the context of 203 CELL BIOLOGY, VOL. 4 Copyright © 1980 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-289504-5 204 Peter J. Roach the functioning of the cell or organism, and particularly, with the role of enzymes in the regulation of cellular processes. The discussion is founded on the assertion that evolution has fashioned the properties of individual proteins to be advantageous for the efficient functioning, and thus survi-val, of the cell or organism, and in this strict biological context, we will talk of the function or purpose of given physical and chemical properties of cellular components (Krebs, 1954; Pittendrigh, 1958; Mayr, 1961; At-kinson, 1970, 1977).

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  The Molecular Fabric of Cells

  • BIOTOL, B C Currell, R C E Dam-Mieras(Authors)
  • 2013(Publication Date)
  • Butterworth-Heinemann

    (Publisher)

  With this information, model-building studies enable the positioning of substrate within the active site to be predicted. In some cases, X-ray diffraction analysis can be carried out with a substrate analogue bound to the active site. These approaches allow a detailed chemical mechanism to be proposed, describing the precise role of the catalytic groups of the enzyme. Detailed descriptions of individual enzyme mechanisms are beyond the scope of this book. We conclude this section by summarising the main ways by which enzymes are believed to achieve catalysis. Note that different enzymes use different ways to achieve rate increases. close contact 8.8.1 Proximity Most reactions involve more than one substrate, although in hydrolytic reactions one of the reactants is the enzyme's solvent, water. For reactions to occur, the two substrates must come close enough (or collide) to react. One way by which enzymes promote reaction is by facilitating the necessary close contact. Since both substrates bind to the enzyme they are brought into close proximity with each other (Figure 8.34). Another way to think of this is that their localised concentration is much higher than it was in free solution. As we have already noted (Section 8.4), chemical rates of reaction increase when substrate concentrations rise. a) in solution, reactants may not collide E Π b) orientation will often be unsatisfactory in free solution -/ s >a c) within the active site substrates are in close proximity and orientation is optimal substrates in active site Figure 8.34 Effect of an enzyme on the proximity and orientation of reactants.

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