Biological Sciences
Measuring enzyme-controlled reactions
Measuring enzyme-controlled reactions involves quantifying the rate at which enzymes catalyze chemical reactions. This is typically done by monitoring changes in substrate concentration, product formation, or enzyme activity over time. Common methods include spectrophotometry, colorimetry, and fluorimetry, which allow for the measurement of enzyme kinetics and the determination of factors affecting enzyme activity.
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8 Key excerpts on "Measuring enzyme-controlled reactions"
- H. Ch. Curtius, Marc Roth(Authors)
- 2018(Publication Date)
- De Gruyter(Publisher)
These units are only meaningful if the enzyme is assayed under zero order kinetics, or under first-order kinetics related to a defined amount of substrate. Incubation conditions should be optimal whenever possible. It is obvious that the existence of several units for the same enzyme can create confusion, especially among clinicians wanting to compare results from different laboratories. It would certainly be more satisfactory if enzymes could be evaluated in terms of micrograms, moles, or active site concentrations (7). Theoretically, this would be possible if reference samples of pure enzymes were available. They could be assayed by any method and serve to establish conversion factors between catalytic units and mass units. This, unfortunately, is hardly possible at present, because few enzymes are commercially available in pure form. Even recrystallized trypsin has been found by active site titration to possess less than 80% of the theoretical catalytic activity (8). Enzymatic Analyses 471 6. Assessment of Reaction Rates Measurement of an enzymatic activity is never instantaneous, as it consists in the determination of a change in substrate or product concentration as a function of time. There are several ways of assessing reaction rates. Continuous recording of the indicator substance concentration versus time is a most reliable method of determining an enzymatic activity. It is feasible if the substance can be selectively and continuously measured under the conditions of incubation. A typical example of such a substance is NADH, which absorbs ultraviolet light and exhibits a blue fluorescence in the pH range where the dehydrogenases to which it is linked are maximally active. Kinetic assay of many hydrolases is made possible by the use of synthetic chromogenic or fluorigenic substrates. For example, alkaline phosphatase hydrolyzes the colorless substrate, p-nitrophenyl phosphate, giving rise to p-nitrophenol, which is yellow in alkaline medium.
eBook - PDFFundamentals of Biochemistry, Student Companion
Life at the Molecular Level
- Akif Uzman, Jerry Johnson, William Widger, Joseph Eichberg, Donald Voet, Judith G. Voet, Charlotte W. Pratt(Authors)
- 2019(Publication Date)
- Wiley(Publisher)
12 Enzyme Kinetics, Inhibition, and Control This chapter introduces chemical kinetics—the study of reaction rates—followed by the kinetics of enzymatic reactions. An enzyme-catalyzed reaction can be described by the Michaelis– Menten equation, which expresses the reaction velocity in terms of its Michaelis constant, K M , and its maximum velocity, V max . Detailed knowledge of the kinetics of a reaction can contribute to the understanding of its step-by-step reaction mechanism. The effects of different substrates, inhibitors, and other factors may also reveal an enzyme’s physiological function. This knowledge can be exploited to develop drugs that are enzyme inhibitors. In this chapter, the three types of reversible enzyme inhibition and the equations that describe them are presented. The chapter also describes the control of enzymes, using aspartate transcarbamoylase as an example of allosteric control, and glycogen phosphorylase as an example of control by covalent modification. The chapter concludes with a discussion of how drugs are developed and tested, and the basis of adverse drug reactions. Essential Concepts Reaction Kinetics 1. A chemical reaction may proceed through several simple steps, called elementary reactions. The overall reaction pathway may therefore involve several short-lived intermediates. 2. The rate, or velocity (v), at which a reactant is consumed or a reaction product appears can be mathematically described. Thus, for the conversion of reactant A to product P, 3. The rate of an elementary reaction varies with the concentration(s) of the reacting molecule(s). For example, for a single-reactant reaction (a unimolecular or first-order reaction), the rate is directly proportional to the concentration of the reactant.
eBook - PDF- Magar Mager(Author)
- 2012(Publication Date)
- Academic Press(Publisher)
CHAPTER 15 RAPID REACTIONS: TRANSIENT ENZYME KINETICS AND OTHER RAPID BIOLOGICAL REACTIONS 1. Introduction In this chapter we concentrate on the determination of kinetic param-eters by techniques generally employed for the study of rapid reactions, namely, flow techniques and relaxation methods. T o borrow a few words from Bernhard (1968), we are talking here of transient kinetics because the turnover number of enzyme catalyzed reactions are rapid (the rate constant for the breakdown of the final enzyme substrate complex before the liberation of products is generally of the order of 10 2 s e c -1 or less). T o investigate what happens during that short time we must employ the special techniques mentioned above. Once we determine the four kinetic parameters [from Eq. (14-17)] for a two substrate reaction studied in the steady state we have exhausted the information available unless we resort to experimental techniques specifically designed to probe the 442 2. Techniques of Measuring Rapid Reactions 443 rapid initial phase of the reaction (Gibson, 1968). Consequently, if one is done with the steady-state kinetics of a reaction and one still wants to do kinetics, then there is no choice but to resort to the measurement of rapid reactions. 2. Techniques of Measuring Rapid Reactions At the heart of the matter, insofar as the techniques for measuring rapid reactions are concerned, lies the measurement of the concentration of various reacting species in as short a time as possible. T o attain this end various investigators have devised different methods. For a survey of the techniques and methods used in chemistry the reader is referred to the volume by Friess et al. (1963) which includes articles by a number of original contributors on the subject: Chance, Roughton, Eigen, DeMayer, Porter, and others. In this volume the authors describe the various instruments (their own and others*) and the methods of measuring rapid reactions.
eBook - PDF- S. Bielecki, J. Polak, J. Tramper(Authors)
- 2000(Publication Date)
- Elsevier Science(Publisher)
The main advantage of the proposed technique is its versatility due to the versatility of the detection principle. The technique can be used, for example, for regular enzyme activity measurement in enzyme inactivation studies or in monitoring of chromatographic enzyme purification. 1. INTRODUCTION An assay for enzyme activity can be arranged generally in two steps. The first one is the enzyme reaction step. Then, atter stopping the reaction, an analytical step is needed for the reactant concentration determination. These two steps can be effectuated simultaneously when one of the reactants can be monitored directly. Changes typically utilized for monitoring enzyme catalyzed reactions are optical properties of the solution, either absorption or emission, concentration of ions, most otten H +, detectable electrochemically. There is a lack of universal methods that allow monitoring of a wider range of enzyme reactions. One of the possible solutions is calorimetry, and it can be used for the kinetic investigation of chemical and enzymatic reactions with a significant heat of reaction. The advantages of kinetic calorimetry are obvious. Firstly, the rate of a chemical reaction can be measured without any special requirements being imposed on the reaction medium (solid, viscous, multicomponent systems). Second, the high efficiency, meaning a large amount of kinetic information from one experiment with a non-destructive character. Third, the chemical conversion is recorded directly at the time of its occurrence [2]. 354 There are two main experimental configurations of calorimetry used for the determination of enzyme activity, i.e. batch [3-7] and flow [8-10]. In this article, a simple procedure based on the flow injection analysis principle is described.
eBook - PDFCell 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).
eBook - ePubBiocatalyst Immobilization
Foundations and Applications
- Maria Lujan Ferreira(Author)
- 2022(Publication Date)
- Academic Press(Publisher)
The efficiency of the immobilization process is then assessed by several parameters. Some consider only the activity registered by a standardized test reaction, while others attempt to quantify the enzyme itself. A myriad of units is then found in the open literature. Boudrant et al. summarized and defined nearly all of them, such as immobilization yield, expressed activity (also called activity recovery), and others, mentioning the problems with calculations too. 13 To these parameters, it is vital to add the enzyme load (EL). Knowing the EL is imperative to compare not only the activities of the free and immobilized enzyme, but also as a comparative parameter between catalysts, and to optimize the design of a biocatalyst in terms of its costs. The calculation of EL is traditionally carried out by the indirect method. That is, by measuring the enzyme concentration in the immobilization solution at the beginning and at the end, as well as in the washings. The immobilized amount is then obtained by difference. Now, how the EL is reported, in which units, and the exact math used to obtain the final reported values can be so different from one laboratory to the other that comparison between biocatalysts is often virtually impossible. This lack of uniformity in criteria may well be one of the causes for the economic potential of biocatalysis being still dismissed by many manufacturers. 6.2.3: A complete analysis of the use of spectrophotometric methods to quantify proteins in biocatalysts and in general As it was presented above practically in all works published over decades, the analytical methods used to determine these enzyme concentrations were originally designed to quantify proteins by the UV-V spectrometry technique
eBook - PDF- Edward Bittar(Author)
- 1996(Publication Date)
- Elsevier Science(Publisher)
A variety of existing approaches provide information relevant to the formulation of biochemical models. The enzymologist' s test tube will continue to provide an indispensable tool in this regard. New tools that will facilitate the measurement of biochemical variables in situ, in a nondestructive fashion, will become increasingly important for characterizing systemic behavior. However, the key to understanding the integrated temporal, spatial and functional behavior of complex biochemical sys- tems will be the development of mathematical formalisms that allow us to relate the molecular and systemic behavior in a deep quantitative manner. The new symbol to represent this emerging paradigm and the indispensable tool for its elaboration will undoubtedly be the integrative biologist's computer. Let us hope that it will serve as long and as well as the enzymologist's test tube. REFERENCES Alberty, R. A. (1959). The rate equation for an enzymatic reaction. In: The Enzymes (Boyer, P. D., Lardy, H., & Myrback, K., eds.), Vol. I, 2nd Ed., pp. 143-155, AcademicPress, New York. Albery, W. J. & Knowles,J. R. (1977). Efficiency and evolution of enzyme catalysis. Angew. Chem. Int. Ed. Engl. 16, 285-293. Bardsley, W.G. & McGinlay, P. B. (1989). Optimal design for model discriminationusing the F-test withnon-linear biochemicalmodels.Criteriafor choosingthe numberand spacingof experimental points. J. Theoret. Biol. 139, 85--102. 142 MICHAEL A. SAVAGEAU Barker, S. A. & Somers, P. J. (1978). Biotechnology of immobilized multienzyme systems. Adv. Biochem. Eng. 10, 27-49. Benson, S. W. (1960). The Foundations of Chemical Kinetics, p. 25, McGraw-Hill, N.Y. Bertalanffy, L. von (1951). Theoretische Biologie, 2nd Ed., Francke, Bern, Switzerland. Benalanffy, L. von (1960). Principles and theory of growth. In: Fundamental Aspects of Normal and Malignant Growth (Nowinski, W. W., ed.), pp. 137-259, Elsevier, N.Y. Bloomfield, V., PeUer, L., & Alberty, R.
- A. Kayode Coker(Author)
- 2001(Publication Date)
- Gulf Professional Publishing(Publisher)
Reactor’s stirrer speed, flowrate, and foaming must be controlled to maintain the productivity of the enzyme. Consequently, during experimental investi-gations of the kinetics enzyme catalyzed reactions, temperature, shear, and pH are carefully controlled; the last by use of buffered solutions. MODELS OF ENZYME KINETICS Consider the reaction S → P occurs with an enzyme as a catalyst. It is assumed that the enzyme E and substrate S combine to form a Biochemical Reaction 835 complex ES, which then dissociates into product P and free (uncombined) enzyme E. E S ES k k + [ 2 1 * (1-93) ES E P k * 3 ⎯ → ⎯ + (1-94) The net rate of disappearance of S is: − ( ) = − ∗ r k C C k C s net E S ES 1 2 (11-1) At pseudo equilibrium (–r s ) = 0, implying that the steps are very rapid: k C C k C E S ES 1 2 = ∗ (11-2) K k k C C C m E S ES = = ∗ 2 1 (11-3) where K m = the dissociation equilibrium constant for ES * C E = concentration of the enzyme, E C S = concentration of the substrate, S C ES * = concentration of the complex, ES * The concentration of the enzyme-substrate complex from Equa-tion 11-3 is C k k C C ES E S ∗ = 1 2 (11-4) Decomposition of the complex to the product and free enzyme is assumed irreversible, and rate controlling: ES P E k ∗ ⎯ → ⎯ + 3 (11-5) The formation rate of the product P is v r k C P ES ≡ ( ) = ∗ 3 (11-6) r r 836 Modeling of Chemical Kinetics and Reactor Design C ES * and C S are related by a material balance on the total amount of enzyme, C ET . C C C E SE ET + = ∗ (1-101) Combining Equations 11-4 and 1-101 gives k k C C C C or ES S ES ET 2 1 ∗ ∗ + = C k C C k k C C C k k C ES S ET S S ET S ∗ = + = + ⎛ ⎝ ⎜ ⎞ ⎠ ⎟ 1 2 1 2 1 (11-7) Combining Equations 11-6 and 11-7 yields v r k C C k k C k C C K C P S ET S S ET m s = ( ) = + ⎛ ⎝ ⎜ ⎞ ⎠ ⎟ = + ( ) 3 2 1 3 (11-8) where K m in this instance is referred to as the Michaelis-Menten [7] constant.
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