Enzyme Kinetics: Catalysis & Control: A Reference of Theory and Best-Practice Methods

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Far more than a comprehensive treatise on initial-rate and fast-reaction kinetics, this one-of-a-kind desk reference places enzyme science in the fuller context of the organic, inorganic, and physical chemical processes occurring within enzyme active sites. Drawing on 2600 references, Enzyme Kinetics: Catalysis & Control develops all the kinetic tools needed to define enzyme catalysis, spanning the entire spectrum (from the basics of chemical kinetics and practical advice on rate measurement, to the very latest work on single-molecule kinetics and mechanoenzyme force generation), while also focusing on the persuasive power of kinetic isotope effects, the design of high-potency drugs, and the behavior of regulatory enzymes.

- Historical analysis of kinetic principles including advanced enzyme science

- Provides both theoretical and practical measurements tools

- Coverage of single molecular kinetics

- Examination of force generation mechanisms

- Discussion of organic and inorganic enzyme reactions

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Editorial Reviews

From the Publisher

"…this is an excellent book. It is difficult to pick it up without finding something new, interesting, and useful. The breadth of coverage and the author’s familiarity with disparate topics are impressive, and the references both to published work and to webbased resources are up to date so that one can jump into the primary literature as necessary."—Catal Lett, 2012, Volume 142 "One of the great strengths of this book is that the voice of the author comes through on every page. He does not simply list different methods for rate equation derivation, for example, but comments on the situations to which each is best suited. Chapter One includes a scolding of the Nobel Prize committee for overlooking Britton Chance’s contributions to science. Critical evaluations of published experimental data and alternative explanations are presented. Articulate arguments for the importance of continued research in enzymology are offered, and there is a thoughtful discussion of how pharmacologically useful compounds are developed. Above all, the people who have conducted the research upon which the book is based are recognized. The list of researchers who ‘‘made advances so notable that they personify the field’’ provides plenty of fodder for late night discussions at the Enzymes Gordon Conference. Whether one agrees with all of Purich’s judgements or not, their inclusion makes reading his book very enjoyable, and also documents his deep familiarity with and affection for the field…this is an excellent book. It is difficult to pick it up without finding something new, interesting, and useful. The breadth of coverage and the author’s familiarity with disparate topics are impressive, and the references both to published work and to web- based resources are up to date so that one can jump into the primary literature as necessary."—Springer, Science and Business Media, LLC

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Product Details

  • ISBN-13: 9780123809247
  • Publisher: Elsevier Science
  • Publication date: 7/6/2010
  • Pages: 920
  • Product dimensions: 8.80 (w) x 11.00 (h) x 2.20 (d)

Meet the Author

Dr. Purich earned his Doctor of Philosophy degree in 1973 for his kinetic characterization of brain hexokinase under the preceptorship of Professor Herbert J. Fromm in the Department of Biochemistry & Biophysics at Iowa State University. As a Staff Research Fellow with Dr. Earl Stadtman at the National Institutes of Health, he conducted research on the cascade of covalent interconverting enzymes that control the glutamine synthetase reaction in Escherichia coli. Dr. Purich subsequently joined the Department of Chemistry at the University of California Santa Barbara where he rose through the ranks as Assistant Professor (1973-78), Associate Professor (1978-82), and Professor (1982-84). At UC Santa Barbara, he was awarded an Alfred P. Sloan Fellow in Chemistry (1978-80), an NIH Research Career Development Award (1978-1983), and the Campus-wide Outstanding Teacher Award (1978). In 1984, he assumed the post of Professor and Chairman of the Department of Biochemistry & Molecular Biology at the University of Florida College of Medicine, and in 1996 he retired as Chairman to resume full-time activities as a professor. Dr. Purich has served on the editorial boards of the Journal of Biological Chemistry (1981-87) and Archives of Biochemistry and Biophysics (1975-85), and as a regular member of the NIH Biochemistry Study Section. Professor Purich succeeds the late Alton Meister as series editor for Advances in Enzymology, and he continues to edit the multi-volume treatise "Enzyme Kinetics & Mechanism" appearing as volumes 63, 64, 87, and 249 in Methods in Enzymology. He has also published Contemporary Enzyme Kinetics and Mechanism (1st ed.,1983; 2nd ed.,1996) based on chapters selected from his Methods volumes.

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Read an Excerpt

Enzyme Kinetics: Catalysis & Control

A Reference of Theory and Best-Practice Methods
By Daniel L. Purich

Academic Press

Copyright © 2010 Elsevier Inc.
All right reserved.

ISBN: 978-0-12-380925-4

Chapter One

An Introduction to Enzyme Science

Enzymes are astonishing catalysts — often achieving rate enhancement factors of 1,000,000,000,000,000,000! Water, electrolytes, physiologic pH, ambient pressure and temperature all conspire to suppress chemical reactivity to such a great extent that even many metabolites as thermodynamically unstable as ATP (ΔGhydrolysis [approximately equal to] -40 kJ/mol) and acetyl-phosphate (ΔGhydrolysis [approximately equal to] -60 kJ/mol) are inert under normal physiologic conditions. Put simply, metabolism would be impossibly slow without enzymes, and Life, as we know it, would be unsustainable. As a consequence, enzymes are virtual on/off- switches, with efficient conversion to products in an enzyme's presence and extremely low or no substrate reactivity in an enzyme's absence. At millimolar concentrations of glucose and MgATP2-, for example, substantial phosphorylation of glucose would require hundreds to thousands of years in the absence of hexokinase, but only seconds at cellular concentrations of this phosphoryl transfer enzyme. Without hexokinase, there would also be no way to assure exclusive phosphorylation at the C-6 hydroxymethyl group. And even when an uncatalyzed reaction (termed the reference reaction) is reasonably fast — as is the case for the reversible hydration of carbon dioxide to form bicarbonate anion or for the spontaneous hydrolysis of many lactones — an enzyme (in this case, carbonic anhydrase) is required to assure that the reaction's pace is compatible with efficient metabolism under the full range of conditions experienced by that enzyme. Most enzymes also exhibit rate-saturation kinetics, meaning that velocity ramps linearly when the substrate concentration is below the Michaelis constant, and reaches maximal activity when the substrate is present at a concentration that is 10–20 times the value of the Michaelis constant. In this respect, an enzyme's action is more akin to a variable-voltage rheostat than a simple on/off switch.

Biochemists recognize that substrate specificity is another fundamental biotic strategy for effectively organizing biochemical reactions into metabolic pathways. Two analogous chemical reactions can take place within the same (or adjoining) subcellular compartments simply because their respective enzymes show substrate or cofactor specificity directing metabolic intermediates to and through their respective pathways, often without any need for subcellular co-localization or enzyme-to-enzyme channeling. Substrate specificity also minimizes formation of unwanted, and potentially harmful, by-products. By controlling the relative concentrations of such enzymes, cells also avoid undesirable kinetic bottlenecks or the undue accumulation of pathway intermediates. Experience tells us that extremely reactive chemical species can also be sequestered within the active sites of those enzymes requiring their formation, while hindering undesirable side-reactions that would otherwise prove to be toxic. So enzyme catalysis is inherently tidy. Enzyme active sites can also harbor metal ions that attain unusually reactive oxidation states that rarely form in aqueous medium and even less often in the absence of side-reactions. The resilience of living organisms stems in large measure from the capacity of enzymes to specifically or selectively bind other ligands (e.g., coenzymes, cofactors, activators, inhibitors, protons and metal ions).

Attesting to the significance of enzyme stereospecificity in the biotic world is that most metabolites and natural products contain one or more asymmetric carbon atoms. The stereospecific action of enzymes is the consequence of the fact that both protein and nucleic acid enzymes are polymers of asymmetric units, making resultant enzymes intrinsically asymmetric. It should be obvious that any L-amino acid-containing polypeptide having even a single D-amino acid residue cannot adopt the same three-dimensional structure as a natural polypeptide. Although some enzymes utilize both enantiomers of a substrate (e.g., glutamine synthetase is almost equally active on D-glutamate and L-glutamate), proteins containing exclusively L-amino acids are produced by the ribosome's peptide-synthesizing machinery. This outcome is the result of the stereospecificity of aminoacyltRNA synthases that supply ribosomes with activated subunits, the stereochemical requirements of peptide synthesis, as well as ubiquitinylating enzymes and proteasomes that respectively recognize and hydrolyze wrongly folded proteins. Cells also produce a range of enzymes, such as D-amino acid oxidase (Reaction: D-Amino Acid + O2 + H2O [??] 2-Oxo Acid + NH3 + H2O2), that remove certain enantiomers (in this case, D-amino acids) from cells. In the case of protein enzymes, certain aspartate residues are also susceptible to spontaneous racemization as well as N-to-O acyl shifts, and cells produce enzymes that recognize and mediate the repair or destruction of proteins containing monomers having improper stereochemistry.

Additional metabolic pathway stability is afforded by steady-state fluxes that resist sudden changes in rate or reactant concentrations. The processes lead to the phenomenon of homeostasis, wherein reactant concentrations appear to be time invariant merely because the processes producing and destroying these reactants are so exquisitely controlled. In some respects, the behavior of the whole of metabolism appears to exceed the sum of behaviors of its individual reactions. Experience has shownthat hierarchically complex, large-scale networks often give rise to emergent properties (i.e., properties of a highly integrated metabolic or physiologic system that are not easily predicted from the analysis of individual components). Beyond the coordinated operation and regulation of the many pathways comprising intermediary metabolism, other emergent properties of living systems are evident in the adaptive resilience of signal transduction, long-range actions affecting chromosomal organization, as well as cellular morphogenesis and motility. The creation of organizationally complex neural networks, as facilitated by the capacity of single neuronal cells to engage in tens of thousands of cell–cell interactions with other neurons via synapse formation, is also thought to underlie what we sense as our own consciousness. And at all such levels, enzyme catalysis and control are inevitably needed for effective intracellular and intercellular communication.

As the essential actuators of metabolism, enzymes are often altered conformationally via biospecific binding interactions with substrates and/or regulatory molecules (known as modulators or effectors) to achieve optimal metabolic control. An additional feature is the capacity of multi-subunit enzymes to exhibit cooperativity (i.e., enhanced or suppressed ligand binding as a consequence of inter-subunit cross-talk). Because enzyme structure changes can be triggered by changes in the concentrations of numerous ligands, enzymes possess an innate capacity to integrate diverse input signals, thereby generating the most appropriate changes in catalytic activity. An interaction is said to be allosteric if binding of a low-molecular weight substance results in a metabolically significant conformational change. In most cases, modulating effects are negative (i.e., they result in inhibition), but positive effects (i.e., those resulting in activation) are also known. Feedback regulation has proven to be a highly effective strategy for controlling the rates of metabolic processes. When present at sufficient concentration, a downstream pathway intermediate or product (known as a feedback inhibitor) alters the structure of its target enzyme to the extent that the inhibited enzyme exhibits little ot no activity (Scheme 1.1). Target enzymes (shown below in red) are most often positioned at the first committed step within a pathway or at a branch point (or node) connecting two or more pathways. The lead reactions are frequently highly favorable (ΔG << 0), whereas the intervening reactions are generally reversible (ΔG = 0), or nearly so (ΔG [approximately equal to] 0).

Feedback inhibition (shown in blue) of target enzymes therefore precludes unnecessary accumulation of possibly toxic metabolic pathway intermediates. By contrast, elevated metabolic throughput (or flux) is observed when an enzyme responds to an allosteric activator. In the latter case, the enzyme achieves no or partial catalytic activity in the absence of an activator, and biospecific binding of the activator alters the target enzyme's conformation in a way that increases its catalytic efficiency. Although the hallmark of allosteric enzymes is cooperativity (i.e., subunit–subunit interactions altering the apparent substrate binding affinity), metabolic control is also achieved by the regulated synthesis and degradation of specific enzymes, by interconversion between enzyme activity states via enzyme-catalyzed covalent modification, by effector molecule mediated signal amplification, and in some instances by substrate channeling.

Molecular life scientists have uncovered countless instances wherein improper catalysis and/or regulation of even a single enzyme reaction can greatly distress a living organism. Such mutant enzymes wreak havoc on cellular physiology. In fact, animal and plant diseases frequently arise from point mutations that result in site-specific substitution of a single amino acid residue in an enzyme. Elaborate proofreading mechanisms permit replication, transcription, and translation to proceed at rapid rates, while minimizing error propagation, and a battery of repair enzymes correct DNA damage arising unavoidably from photolysis, oxidation, alkylation, hydrolysis, and racemization. The same is true of errors occurring during the synthesis, splicing, and turnover of RNA transcripts. Ribosomes must also occasionally commit errors, but with the possible exception of prion protein formation the impact of low-level occurrence of "translational mutations" is apt to be minimal. Other more injurious mistakes made during replication and transcription are known to culminate in enzyme over-/under-production, defective regulation, impaired stability, incorrect post-translational modification, improper subcellular targeting and compartmentalization, defective turnover, etc. A notable example is amyotrophic lateral sclerosis or ALS (widely known as Lou Gehrig's disease). This devastating neurodegenerative disorder is linked to the impaired action of superoxide dismutase; overaccumulation of superoxide ([MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]) damages neurons, an injury that is attended by profound pathological sequelae. Another example is the discovery that Pin1-catalyzed cis-trans prolyl residues isomerization can alter the structure of the microtubule-associated protein Tau in axons and that Pin1 gene knockouts bring about progressive age dependent neuropathy characterized by motor and behavioral deficits, attended by hyper-phosphorylation of Tau, as well as Tau polymerization into neurodegenerative paired helical filaments (Liou et al., 2003). Although more research is required to assess the significance of such findings to the onset of Alzheimer's disease, it is already clear that reduced prolyl cis-trans isomerization activity can profoundly impair neuronal function.

Enzyme chemists investigate biological catalysis by assessing the structural and energetic features of the elementary reactions comprising a multi-step enzyme mechanism. They seek to understand how activators and inhibitors alter the energetics of catalytic reaction cycles to bring about effective metabolic regulation. The daunting task of determining how an enzyme operates is never an easy matter, and without a systematic approach, one is forced to glean information haphazardly. A more effective strategy starts with a reliable assay of catalytic activity and requires the experimenter to use this assay in the isolation of the enzyme of interest from other contaminants (e.g., proteins, solutes, etc.) affecting the enzyme's activity. In practice, absolute purity is not required as long as other contaminating enzymes and proteins are without effect on the enzyme of interest. It is helpful to apply the principles of organic chemistry to infer likely chemical transformations occurring during catalysis, using literature precedents to guide one's thoughts about the roles of coenzymes and cofactors and to focus on probable reaction intermediates. Ultimately, however, it is necessary to test whether each reaction step occurs on a time-scale consistent with its role in catalysis. This latter pursuit, called enzyme kinetics, combines an interest analyzing temporal aspects of enzyme catalysis with the principles of physical chemistry and quantitative rigor of analytical chemistry.

Some of the stages in the characterization of a complete enzyme mechanism are listed in Fig. 1.1. Because initial-rate kinetics is a relatively straightforward tool for analyzing enzyme catalysis, we may regard such experimental approaches as the first stage in the systematic characterization of an enzyme of interest. Pursuit of subsequent stages depends on the objectives of the particular investigation.

This reference explains how enzyme kineticists formulate and test models to: (a) explain the reactivity and energetics of enzyme processes; (b) gain the most complete description of catalysis; and (c) understand how an enzyme's regulatory interactions affect the catalytic reaction cycle. Ideally, one should consider as many reasonable models as possible for the reaction/process of interest. These rival kinetic models should be as simple as possible: when stripped down to the bare essentials, any failure of a model to account for an experimentally determined property of the system becomes sufficient justification for outright rejection of that model or for modifying it to account for other by essential properties/ interactions. Simplicity, precision, and generativity — these are the inherent virtues of highly effective models. Simplicity demands that a system's known properties are represented by the least number of components and/or interactions. Precision requires explicit presentation of all required interactions, thus providing an opportunity to distinguish testable model-specific characteristics of rival models. Generativity implies that the model should facilitate hypothesis-driven experimentation to test newly predicted properties in a recursive manner that stimulates new rounds of experimentation. Put plainly, a model is not worth much, unless it fosters the formulation of new hypotheses that spur additional rounds of experimentation.

Modern molecular life scientists have become, for want of a more appropriate appellation, "interaction spectroscopists" — focusing on the spectrum of interactions of proteins and enzymes with other proteins, nucleic acids, membranes, and low molecular-weight metabolites, most often in terms of location, specificity, affinity, and catalysis. And because enzymes are Life's actuators, it should not be surprising that, whenever a significant problem in the molecular life sciences reaches a sophisticated level of understanding, an enzyme is almost invariably involved. Because all kinetic approaches are fundamentally similar, those gaining mastery over the topics presented in this book can become proficient at inventing their own kinetic approaches for testing their own models. Moreover, because biochemical principles underlie the entirety of the molecular life sciences, these strategies should also be useful for investigators seeking to unravel the time-ordered events of highly complex biotic processes in the fields of molecular and cell biology, physiology and neuroscience, as well as microbiology and the plant sciences.


Excerpted from Enzyme Kinetics: Catalysis & Control by Daniel L. Purich Copyright © 2010 by Elsevier Inc.. Excerpted by permission of Academic Press. All rights reserved. No part of this excerpt may be reproduced or reprinted without permission in writing from the publisher.
Excerpts are provided by Dial-A-Book Inc. solely for the personal use of visitors to this web site.

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Table of Contents

  1. An Introduction to Enzyme Rate Processes
  2. Active Sites & Their Chemical Properties
  3. Fundamentals of Chemical Kinetics
  4. Practical Aspects of Measuring Rates & Kinetic
  5. Initial-Rate Kinetics of One-Substrate Enzyme-Catalyzed Reactions
  6. Initial-Rate Kinetics of Multi-Substrate Enzyme-Catalyzed Reactions
  7. Other Factors Influencing Enzyme Activity
  8. Kinetic Behavior of Enzyme Inhibitors
  9. Isotopic Probes of Biological Catalysis
  10. Probing Fast Enzyme Processes
  11. Regulatory Behavior of Enzymes
  12. Single-Molecule Enzyme Kinetics
  13. Force Generation in Mechanoenzyme Catalysis
  14. On Keeping a Research Notebook
  15. Steady-State Rate Equations for Selected One- & Two-Substrate Enzyme-Catalyzed Reactions
  16. Summary of Enzyme Kinetic Mechanisms Abbreviations & Symbols References
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