ENZYMES: Catalysis, Kinetics and Mechanisms 2018 PDF

N. S. Punekar ENZYMES: Catalysis, Kinetics and Mechanisms ENZYMES: Catalysis, Kinetics and Mechanisms N. S. Punekar ENZYMES: Catalysis, Kinetics and Mechanisms N. S. Punekar Department of Biosciences & Bioengineering Indian Institute of Technology Bombay Mumbai, Maharashtra, India ISBN 978-981-13-0784-3 ISBN 978-981-13-0785-0 (eBook) https://doi.org/10.1007/978-981-13-0785-0 Library of Congress Control Number: 2018947307 # Springer Nature Singapore Pte Ltd. 2018 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, speciﬁcally the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microﬁlms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a speciﬁc statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. Printed on acid-free paper This Springer imprint is published by the registered company Springer Nature Singapore Pte Ltd. The registered company address is: 152 Beach Road, #21-01/04 Gateway East, Singapore 189721, Singapore For Sandhya, Jahnavi, and Chaitanya Preface Any living being is a reﬂection of its enzyme arsenal. We are and do what our enzymes permit. Christian de Duve Enzymes are the lead actors in the drama of life. Without these molecular machines the genetic information stored in DNA is worthless. With rising attention to the fashionable ﬁelds like molecular biology, genetic engineering, and biotech- nology, the techniques to manipulate DNA have occupied center stage. Being popular, many concepts of molecular biology/genetic engineering are now introduced to undergraduates. Unfortunately, this has happened at the cost of other fundamental facets of biology, including enzymology. In the excitement to collate volumes of data for Systems Biology (and the various “Omics” fashions), the beauty and vigor of careful analysis – one enzyme at a time – is neglected. It is an intellectual challenge to assay individual enzymes while avoiding complications due to others – an almost forgotten activity in modern biology. Many in the present generation assume that performing one standard assay will tell you everything about that enzyme. While biochemists spent lifetimes on a single native enzyme, the notion today is that one can characterize a mutant in the morning! Over the last three decades devoted enzymologists have become a rare breed. Many Biology teaching programs have expanded in the areas of molecular and cellular biology while they manage with a makeshift enzymology instructor. New students who are attracted to the study of enzymes do exist, but they ﬁnd themselves in a very bleak teaching environment. Not surprisingly their numbers are dwindling. Reservoirs that are not replenished may soon run dry. Purpose of This Book Genes for enzymes are routinely ﬁshed out, cloned, sequenced, mutated, and expressed in a suitable host. Characterizing the mutant enzyme, however, requires a thorough mechanistic study – both chemical and kinetic. It is thus an exciting time to do enzymology. Hopefully, this book provides enough basic exposure to make this happen. vii viii Preface The ease with which sophisticated data are collected nowadays has dispirited the slow and burdensome approach of resolving and reconstituting a complex enzyme system. Micro-arrays that measure the transcription of many genes at a time disclose neither the abundance nor any attributes of the enzymes/proteins they encode. As F.G. Hopkins wrote in 1931 “..the biochemist’s word may not be the last in describing life, but without his help, the last word will never be said.” This is true of enzymology as well. While the interest and expertise in teaching/learning enzy- mology has declined exponentially, working knowledge of enzymology remains indispensable. Enzymes have come to occupy vast areas of modern biology research and the biotechnology industry. Enzymes whether used as popular kits, mere research tools, or for their own sake require a minimal appreciation of their workings. A tome on enzymology that focuses and logically connects theory of enzyme action to actual experimentation is desirable. One objective of this book is to bridge this gap and enable students to understand, design, and execute enzyme experiments on their own. Enzyme study can range from the simple to the most complicated. Approaches that can be performed in a modest laboratory setup and with no fancy equipment are needed. Conveying the excitement of enzymology within a modest budget and with few experiments is desirable. And hence, equipment intensive approaches – such as structural enzymology, sophisticated techniques like X-ray, NMR, ESR, fast reactions, and isotope effects – have received a somewhat limited coverage. Readers interested in them will yet ﬁnd sufﬁcient background material here. Audience and Their Background Reasons for the cursory coverage of enzymology in most contemporary biology academic programs are twofold. Over-emphasis and glamorization of molecular biology (later genetic engineering!) in the last few decades has captured a dispropor- tionately large allocation of resources and time. Secondly, as a cumulative effect of this attitude, very few well-trained specialists in enzymology are available today. Therefore, study material that encourages students/researchers to understand, design, and execute experiments involving enzymes on their own is needed. The contents of the present book are expected to serve this purpose. Most biochemistry and molecular biology students are introduced to enzymes as commercial reagents and as faceless as buffers and salts. This has led to inadequate appreciation of enzymology and its practices. Standards for reporting enzymology data (STRENDA; available at http://www.strenda-db.org) are a recent effort to prescribe the best approaches to generate and report enzyme data. With an ever- increasing reliance on genomics and proteomics, enzymes are no longer isolated and/or assayed for activity. Often their role is inferred from sequence data alone. “Molecular biology falters when it ignores the chemistry of the products of DNA blueprint – enzymes – the protein catalysts of the cellular machinery.” This philoso- phy was beautifully reiterated by Arthur Kornberg in his “Ten Commandments of Enzymology” (J Bacteriol. (2000) 182:3613–3618; TIBS (2003) 28:515–517). The Preface ix present book is an attempt to sift through chemical sophistication and simplify it for an audience with a biology background. It will serve the curricular needs of senior undergraduates and postgraduates in Biochemistry, Biotechnology, and most branches of modern biology. Dealing with reaction rates, enzymology is a quantitative and analytical facet of biological understanding. Appreciation of rate equations and their meaning therefore becomes important. Minimal competence with algebra, logarithms, exponential relationships, equations to ﬁt straight lines, and simple curves is crucial. While one need not be scared of fearsome equations, the essence of the physical models they represent (or do not represent!) ought to be understood. To an extent, this book is my response to oust the fear of the quantitative in the students of Biology. Because enzymes catalyze chemical reactions, chemical mechanisms are of great concern. They are best understood with adequate preparation in concepts like valency, movement of electrons and charges in molecules, acids and bases, etc. The study of mechanistic enzymology is meaningless without this background. We may recall from Emil Fischer’s Faraday Lecture to the Chemical Society in 1907: “... the separation of chemistry from biology was necessary while experimental methods and theories were being developed. Now that our science is provided with a powerful armoury of analytical and synthetic weapons, chemistry can once again renew the alliance with biology, not only for the advantage of biology but also for the glory of chemistry.” Enzymology without Chemistry (physical and organic) is a limited descriptor of surface (superﬁcial!) phenomena. This requirement obviously puts some burden on students who have lost touch with chemistry for few years in the pursuit of “Biology Only” programs. Basic knowledge on amino acids, their reactivity, and protein structure is a prerequisite to study enzymes. Protein (and hence enzyme) puriﬁcation methods/ tools like various fractionation/separation techniques and chromatographies are not explicitly covered here. Also, essential techniques of protein structure determination do not ﬁnd a dedicated treatment in this book. One may ﬁnd such background material in the standard text books of biochemistry. Lastly, the reader is expected to be familiar with the concepts of concentrations, ionic strength, pH, etc. and exposure to biochemical calculations is essential. Organization This book endeavors to synthesize the two broad mechanistic facets of enzymology, namely, the chemical and the kinetic. It also attempts to bring out the synergy between enzyme structures and mechanisms. Written with self study format in mind, the emphasis is on how to begin experiments with an enzyme and subse- quently analyze the data collected. Individual concepts are treated as stand-alone short sections, and the book is largely modular in organization. The reader can focus on a concept (with real examples) with minimal cross-referencing to the rest of the book. Many attractive enzymes were consciously passed up in order to suit the “Biology” audience. This error of omission painfully belongs to the author. A x Preface limited treatment on applied aspects of enzymes is deliberate as one fully subscribes to Louis Pasteur’s dictum – “There are no applied sciences....The study of the applications of science is easy to anyone who is master of the theory of it.” The book then would also have become unmanageably long. Individual concepts (as chapters) are conveniently grouped into ﬁve broad parts. It all begins with an overview of enzyme catalysis (Part I) followed by a section (Part II) on kinetic practices and measurement of enzyme activity. Two major themes of mechanistic enzymology, namely, the kinetic (Part III) and the chemical (Part IV) occupy bulk attention. A short piece on integrating enzyme kinetic and chemical mechanisms (in Part IV) is a novelty and should be of value. Aspects of enzymology in vivo and frontier research themes form the last section (Part V). The original literature for this book was collected up to year 2016. Fresh research material, constantly being added to many topics, made it hard to draw this boundary. Otherwise, the book would have been always under preparation! Besides listing select text books and original publications, references to recent reviews on most topics are provided. Wherever possible, literature is cited from easily available and open-access resources. How to Use This Book The book contains a balance of physical and chemical fundamentals. Students of modern biology come from many different backgrounds. Hopefully, those from more physical and chemical background will enjoy the material as is. Many of the physico-chemical concepts and mathematical material may be difﬁcult to students narrowly exposed to biological sciences alone. The essential theory to help such audience is presented in Chaps. 9 and 10 (covering chemical kinetics) and 29, 30, and 31 (covering organic reaction mechanisms). It is highly recommended that the uninitiated read these chapters ﬁrst. Chapter 24 arrives before a primer on acid-base chemistry in Part IV; hence, it is suggested to read Chap. 30 before approaching the material in Chap. 24. A complete mechanistic understanding of enzyme action is possible only through a variety of experimental approaches. How these bits of information are combined to arrive at the ﬁnal description may be found in Chaps. 28 and 36. Inclusion of regulation of enzyme activity (Chap. 37) under Frontiers of Enzymology (in Part V) may not be such a revelation since novel regulatory features are being discovered with remarkable regularity. Mumbai, Maharashtra, India N. S. Punekar Useful Constants and Conversion Factors Calorie (cal): (Heat required for raising the temperature of 1 g water from 14.5 C to 15.5 C) 1 cal ¼ 4.184 J 1 kcal ¼ 1000 cal ¼ 4184 J Joule (J): 1 J ¼ 0.239 cal ¼ 1 kg m2 s2 ¼ 2.624 1019 eV Coulomb (C): 1 C ¼ 6.242 1018 electron charges Avogadro’s number (N): N ¼ 6.022 1023 mol 1 Faraday constant (F): 1 1 1 F ¼ 23.063 kcal V mol ¼ N electron charges ¼ 96,480 C mol Boltzmann constant (kB): kB ¼ 1.381 10 23 J K 1 ¼ 1.38 10 16 cm2 g s 2 K 1 Plank’s constant (h): h ¼ 6.626 10 34 J s ¼ 6.626 10 27 cm2 g s 1 Gas constant (R): 1 1 1 1 R ¼ N kB ¼ 1.987 cal mol K ¼ 8.315 J mol K Absolute temperature (degree Kelvin, K): 0 K ¼ absolute zero ¼ 273 C; 25 C ¼ 298 K RT at 25 C: 1 1 RT ¼ 2.478 kJ mol ¼ 0.592 kcal mol xi xii Useful Constants and Conversion Factors Units for ΔG, ΔH, and ΔS: For ΔG and ΔH: cal mol 1 (or J mol 1) For ΔS: calmol 1 K 1 (or J mol 1 K 1) Enzyme catalytic unit: 1 U ¼ 1 μmol min 1 ¼ 16.67 nkatal 1 katal ¼ 1 mol s 1 Curie (Ci): Quantity of a radioactive substance that decays at a rate of 2.22 1012 disintegrations per minute (dpm) Acknowledgments In the era of molecular biology, genetic engineering, and genomics, enzymology is often deemed unglamorous. In this backdrop, it is my good fortune to have beneﬁted from the wisdom of a few enzymology stalwarts. I was initiated into research on enzymes at the Indian Institute of Science, Bangalore. But after a stint at Institute for Enzyme Research, UW-Madison, I was consumed by this evergreen subject. I owe much to these two great institutions in whetting my appetite for enzymology and the preparation for this book. Being a postdoctoral fellow in Prof. Henry Lardy’s group and taking a course on enzymes with Prof. WW Cleland were invaluable. Much of the ground covered in this book was developed while teaching the “Molecular Enzymology” course at IIT Bombay, over 25 years. It was exciting to teach and learn about enzymes with so many bright and committed graduate students. Those indifferent to enzymology (and there were many!) helped me evolve a few tricks to get them interested – I am grateful to them. Any good feature of this book is clearly a result of such an exposure. I thank my colleagues in the department, particularly, Professors K.K. Rao, P.J. Bhat, and P.V. Balaji, who presumed my competence in the subject; this pushed me to exert more and do better. Thanks to Prof. P Bhaumik for enriching me with the structural aspects of enzymology. The material and the organization of this book evolved over the years. The work was initiated during 2007 while on sabbatical leave from IIT Bombay. The ﬁnancial support for book writing from Continuing Education Program (CEP) cell at IIT Bombay is gratefully acknowledged. The inputs of four anonymous reviewers improved the quality of this book and for this I am indebted to them. Encouragement and generous support of Ms. Suvira Srivastav, Dr. Bhavik Sawhney, and Ms. Saanthi Shankhararaman from Springer Nature was valuable in bringing this book to fruition. Salvador Dalí once said – “Have no fear of perfection – you’ll never reach it.” Surely, this book has its own share of glitches. All those errors and limitations are mine alone; I will be very grateful to the readers for pointing them out to me ([email protected]) for rectiﬁcation. This book would not have been possible without the academic spirit inculcated in me by my father. I am deeply indebted to three women for inspiration – my mother xiii xiv Acknowledgments (Akka) for always believing in me, my wife (Sandhya) for the constant reminder that in the race for quality there is no ﬁnish line, and my daughter (Jahnavi) for allowing me to dream even the impossible. I particularly thank my wife Sandhya for her continued support during the longer than anticipated gestation period of this book. Contents Part I Enzyme Catalysis – A Perspective 1 Enzymes: Their Place in Biology........................... 3 Suggested Reading...................................... 4 2 Enzymes: Historical Aspects.............................. 5 2.1 Biocatalysis: The Beginnings.......................... 5 2.2 “Enzyme”: Conceptual Origin......................... 7 2.3 Key Developments in Enzymology..................... 8 Reference............................................. 13 3 Exploiting Enzymes: Technology and Applications............. 15 3.1 Exploiting Natural Diversity.......................... 16 3.2 Modifying Enzymes to Suit Requirements................ 22 3.3 Genetic Engineering and Enzymes...................... 27 3.4 Summing Up..................................... 30 References............................................ 30 4 On Enzyme Nomenclature and Classiﬁcation................. 33 4.1 What Is in the Name?............................... 33 4.2 Enzyme Diversity and Need for Systematics............... 34 4.3 Enzyme Commission: Recommendations................. 35 4.4 Some Concerns.................................... 39 References............................................ 41 5 Hallmarks of an Enzyme Catalyst.......................... 43 5.1 Catalysis........................................ 43 5.2 Speciﬁcity....................................... 46 5.3 Regulation....................................... 49 References............................................ 51 6 Origins of Enzyme Catalytic Power......................... 53 6.1 Proximity and Orientation Effects...................... 53 6.2 Contribution by Electrostatics......................... 57 6.3 Metal Ions in Catalysis.............................. 60 6.4 General Acid–Base Catalysis.......................... 62 xv xvi Contents 6.5 Covalent Catalysis................................. 64 6.6 Transition State Binding and Stabilization................. 65 References............................................ 69 7 Which Enzyme Uses What Tricks?........................ 71 References............................................ 74 8 Structure and Catalysis: Conformational Flexibility and Protein Motion.............................................. 75 References............................................ 82 Part II Enzyme Kinetic Practice and Measurements 9 Chemical Kinetics: Fundamentals.......................... 85 9.1 Measurement of Reaction Rates........................ 85 9.2 Factors that Inﬂuence Chemical Reaction Rates............. 87 9.3 Reaction Progress and Its Concentration Dependence........ 87 9.4 Temperature Dependence of Reaction Rates............... 91 9.5 Catalysis........................................ 94 9.6 Purpose of Kinetic Studies: Reaction Mechanism........... 94 Reference............................................. 96 10 Concepts of Equilibrium and Steady State................... 97 10.1 Chemical Reaction Equilibrium........................ 98 10.2 Binding Equilibrium................................ 102 10.3 Complex Reactions Involving Intermediates............... 103 References............................................ 106 11 ES Complex and Pre-steady-state Kinetics................... 107 11.1 ES Complex, Intermediates, and Transient Species.......... 108 11.2 Kinetic Competence of an Intermediate.................. 110 11.3 Pre-steady-state Kinetics............................. 110 References............................................ 114 12 Principles of Enzyme Assays.............................. 115 12.1 Detection and Estimation Methods...................... 115 12.2 Enzyme Reaction Time Course........................ 120 12.3 Precautions and Practical Considerations................. 123 12.4 Summing Up..................................... 127 References............................................ 129 13 Good Kinetic Practices.................................. 131 13.1 How to Assemble Enzyme Assay Mixtures................ 131 13.2 pH and Ionic Strength Considerations.................... 137 13.3 Temperature Effects................................ 139 13.4 Summing Up..................................... 141 References............................................ 142 Contents xvii 14 Quantiﬁcation of Catalysis and Measures of Enzyme Purity...... 143 14.1 Enzyme Units, Speciﬁc Activity, and Turnover Number...... 143 14.2 Enzyme Puriﬁcation and Characterization................. 146 14.3 Interpreting a Puriﬁcation Table: Criteria of Enzyme Purity.... 148 14.4 Unity of the Enzyme................................ 150 14.5 Summing Up..................................... 153 References............................................ 153 15 Henri–Michaelis–Menten Equation......................... 155 15.1 Derivation of the Michaelis–Menten Equation.............. 155 15.2 Salient Features of Michaelis-Menten Equation............. 159 15.3 Signiﬁcance of KM, Vmax, and kcat/KM................... 165 15.4 Haldane Relationship: Equilibrium Constant Meets Kinetic Constants........................................ 171 15.5 Use and Misuse of Michaelis–Menten Equation............ 175 References............................................ 175 16 More Complex Rate Expressions........................... 177 16.1 Investigating Enzyme Mechanisms Through Kinetics........ 177 16.2 Notations and Nomenclature in Enzyme Kinetics........... 179 16.3 Deriving Rate Equations for Complex Equilibria............ 182 16.3.1 Algebraic Method........................... 182 16.3.2 King–Altman Procedure....................... 184 16.3.3 Net Rate Constant Method..................... 187 16.3.4 Other Methods.............................. 190 16.4 Enzyme Kinetics and Common Sense.................... 190 References............................................ 191 17 Enzyme Kinetic Data: Collection and Analysis................ 193 17.1 Obtaining Primary Data: Practical Aspects................ 193 17.1.1 Reductionism in Experimental Design............. 193 17.1.2 Choice of Substrate Concentrations............... 194 17.1.3 Pilot Experiments and Iteration.................. 195 17.1.4 Importance of Measuring Initial Velocities.......... 196 17.1.5 Utility of the Integrated Form of Michaelis–Menten Equation.................................. 197 17.2 Analyzing Data: The Basics........................... 198 17.2.1 Variation, Errors, and Statistics.................. 198 17.3 Plotting v Versus [S] Data............................ 199 17.3.1 The v Versus [S] Plot......................... 199 17.3.2 Direct Linear Plot............................ 200 17.3.3 v Versus log[S] Plot.......................... 201 17.3.4 Hill Plot.................................. 203 xviii Contents 17.4 Linear Transforms of Michaelis–Menten Equation........... 204 17.4.1 Lineweaver–Burk Plot........................ 205 17.4.2 Eadie–Hofstee Plot........................... 208 17.4.3 Woolf–Hanes Plot........................... 209 17.5 Summing Up..................................... 211 References............................................ 211 Part III Elucidation of Kinetic Mechanisms 18 Approaches to Kinetic Mechanism: An Overview.............. 215 18.1 Which Study Gives What Kind of Information?............ 216 18.2 Two Thumb Rules................................. 217 19 Analysis of Initial Velocity Patterns......................... 221 19.1 Intersecting Patterns................................ 222 19.1.1 Determination/Evaluation of Kinetic Constants and Replots................................... 222 19.1.2 Interpretation............................... 224 19.2 Parallel Patterns................................... 225 19.2.1 Determination/Evaluation of Kinetic Constants and Replots................................... 226 19.2.2 Interpretation............................... 226 19.3 Few Unique Variations.............................. 228 Appendix............................................. 229 References............................................ 230 20 Enzyme Inhibition Analyses.............................. 231 20.1 Reversible Versus Irreversible Inhibition................. 231 20.2 Partial Versus Complete Inhibition...................... 233 20.3 Other Inhibitor Types............................... 234 References............................................ 236 21 Irreversible Inhibitions.................................. 237 21.1 Chemical Modiﬁcation Agents......................... 237 21.2 Afﬁnity Labels.................................... 241 21.3 Suicide Substrates.................................. 242 21.4 Tight-Binding Inhibitors............................. 243 References............................................ 244 22 Reversible Inhibitions................................... 245 22.1 Competitive Inhibition............................... 246 22.1.1 Determination/Evaluation of Kinetic Constants and Replots................................... 247 22.1.2 Interpretation............................... 248 22.2 Uncompetitive Inhibition............................. 248 22.2.1 Determination/Evaluation of Kinetic Constants and Replots................................... 249 22.2.2 Interpretation............................... 250 Contents xix 22.3 Noncompetitive Inhibition............................ 250 22.3.1 Determination/Evaluation of Kinetic Constants and Replots................................... 251 22.3.2 Interpretation............................... 252 22.4 Reversible Inhibition Equilibria: Another Viewpoint......... 253 22.4.1 Signiﬁcance of α and β Values.................. 254 22.5 IC50 and Its Relation to KI of an Inhibitor................. 254 Appendix............................................. 256 References............................................ 258 23 Alternate Substrate (Product) Interactions................... 259 23.1 Substrate Inhibition................................. 259 23.1.1 Determination of Kinetic Constants and Their Signiﬁcance................................ 260 23.2 Use of Alternate Substrates in Enzyme Studies............. 261 23.2.1 Information About the Active Site Shape, Geometry, and Interactions............................. 262 23.2.2 Understanding Kinetic Mechanism............... 266 Reference............................................. 266 24 pH Studies with Enzymes................................ 267 24.1 Enzyme pH Optimum............................... 268 24.2 pH Kinetic Proﬁles................................. 269 24.3 Identifying Groups Seen in pH Proﬁles................... 272 Reference............................................. 274 25 Isotopes in Enzymology.................................. 275 25.1 Enzyme Assays with a Radiolabeled Substrate............. 276 25.2 Isotope Partitioning................................. 277 References............................................ 279 26 Isotope Exchanges at Equilibrium.......................... 281 26.1 Partial Reactions and Ping-Pong Mechanism............... 282 26.2 Sequential Mechanisms.............................. 283 References............................................ 286 27 Isotope Effects in Enzymology............................. 287 27.1 Magnitude of the Observed Isotope Effect................ 289 27.2 Experimental Approaches to Measure Isotope Effects........ 292 27.2.1 Direct Comparison........................... 292 27.2.2 Equilibrium Perturbation....................... 293 27.2.3 Internal Competition Method................... 293 27.3 Applications of KIEs in Enzymology:................... 294 27.3.1 Elucidating Kinetic Mechanism.................. 294 27.3.2 Deciding Chemical Mechanism.................. 294 27.3.3 Understanding Enzyme Transition State............ 297 References............................................ 299 xx Contents 28 From Kinetic Data to Mechanism and Back.................. 301 28.1 How to Relate Mechanisms with Steady-State Kinetic Data.... 301 28.1.1 Ordered Mechanism.......................... 302 28.1.2 Random Mechanism.......................... 302 28.1.3 Ping-Pong Mechanism........................ 305 28.2 Assigning Kinetic Mechanisms: An Action Plan............ 306 28.3 Practical Relevance of Enzyme Kinetics.................. 307 28.3.1 Afﬁnity Chromatography and Protein Puriﬁcation.... 307 28.3.2 Dissection of Metabolism...................... 308 28.3.3 Enzyme–Targeted Drugs in Medicine............. 308 References............................................ 310 Part IV Chemical Mechanisms and Catalysis 29 Chemical Reactivity and Molecular Interactions............... 313 29.1 Atoms, Molecules, and Chemical Bonding................ 313 29.1.1 Covalent Bonds............................. 314 29.1.2 Directional Property of Covalent Bonds............ 316 29.1.3 Non–covalent Interactions and Intermolecular Forces.................................... 318 29.2 Chemical Reaction Mechanisms........................ 320 29.2.1 Cleaving and Forming Covalent Bonds............ 320 29.2.2 Logic of Pushing Electrons and Moving Bonds...... 322 29.3 Stereochemical Course of Reaction..................... 324 29.4 Common Organic Reaction Types...................... 325 29.4.1 Nucleophilic Displacements.................... 326 29.4.2 Elimination Reactions......................... 327 29.4.3 Carbon–Carbon Bond Formation................. 328 29.5 Summing Up..................................... 330 Reference............................................. 330 30 Acid–Base Chemistry and Catalysis........................ 331 30.1 Acids and Bases................................... 331 30.2 General Acid–Base Catalysis.......................... 338 30.3 Summing Up..................................... 342 References............................................ 343 31 Nucleophilic Catalysis and Covalent Reaction Intermediates...... 345 31.1 Nucleophiles and Electrophiles Available on the Enzyme..... 345 31.2 Nucleophilic (Covalent) Catalysis...................... 350 31.3 Covalent Reaction Intermediates....................... 355 31.4 Detecting Intermediates and Establishing Their Catalytic Competence...................................... 357 31.5 Summing Up..................................... 364 References............................................ 365 Contents xxi 32 Phosphoryl Group Chemistry and Importance of ATP.......... 367 32.1 Why Nature Chose Phosphates........................ 367 32.2 Chemical Mechanisms at the Phosphoryl Group............ 368 32.3 Adenosine Triphosphate: Structure Relates to Function....... 373 32.4 Investing Group Transfer Potential to Create Good Leaving Groups.......................................... 380 32.5 Summing Up..................................... 382 References............................................ 383 33 Enzymatic Oxidation–Reduction Reactions................... 385 33.1 What Are Oxidation–Reduction Reactions?............... 385 33.2 How Enzymes Inﬂuence Redox Reaction Rates............. 390 33.3 Mechanisms and Modes of Electron Transfer.............. 392 33.4 Pterine and Folate Cofactors.......................... 393 33.5 Nicotinamide Cofactors.............................. 394 33.6 Flavins and Flavoenzymes............................ 396 33.7 Reactions Involving Molecular Oxygen.................. 398 33.8 Summing Up..................................... 401 References............................................ 402 34 Carboxylations and Decarboxylations....................... 403 34.1 Reactions and Reactivity of CO2....................... 403 34.2 Carboxylation Chemistry with Pyruvate and Phosphoenolpyruvate............................... 405 34.3 Cofactor-Assisted Carboxylations....................... 407 34.4 Decarboxylation Reactions........................... 412 34.5 Thiamine Pyrophosphate and α-Keto Acid Decarboxylations... 415 34.6 Summing Up..................................... 419 References............................................ 420 35 Electrophilic Catalysis and Amino Acid Transformations........ 421 35.1 Protein Electrophiles................................ 423 35.2 Reactions Involving Pyridoxal Phosphate (PLP)............ 428 35.3 Summing Up..................................... 433 References............................................ 436 36 Integrating Kinetic and Chemical Mechanisms: A Synthesis...... 437 36.1 Competence of the Proposed Reaction Intermediate.......... 437 36.2 Glutamine Synthetase............................... 439 36.3 Glutamate Dehydrogenase............................ 442 36.4 Disaccharide Phosphorylases.......................... 443 36.5 Acyl Transferases.................................. 446 36.6 Chymotrypsin..................................... 448 xxii Contents 36.7 Aldolases and Transaldolase.......................... 450 36.8 Ribonuclease A.................................... 454 36.9 Interdependence of Kinetic and Chemical Mechanisms: A Summary...................................... 455 References............................................ 457 Part V Frontiers in Enzymology 37 Regulation of Enzyme Activity............................ 461 37.1 Control of Enzyme Concentration...................... 463 37.2 Control of Enzyme Activity: Inhibition................... 465 37.3 Control of Enzyme Activity: Cooperativity and Allostery..... 468 37.4 Isozymes and Regulation............................. 475 37.5 Covalent Modiﬁcations and Control..................... 479 37.6 Protein-Protein Interactions and Enzyme Control............ 484 37.7 Compartmental Regulation and Membrane Transport........ 485 37.8 Glutamine Synthetase: An Anthology of Control Mechanisms...................................... 488 37.9 Summing Up..................................... 490 References............................................ 492 38 In Vitro Versus In Vivo: Concepts and Consequences........... 493 38.1 Why Michaelis-Menten Formalism Is Not Suitable In Vivo.... 494 38.2 Concentration of Enzymes, Substrates, and Their Equilibria.... 497 38.3 Avogadro’s Number Is a Very Big Number............... 500 38.4 Diffusion, Crowding, and Enzyme Efﬁciency.............. 504 38.5 Consecutive Reactions and Metabolite Channeling.......... 510 38.6 Summing Up..................................... 517 References............................................ 518 39 Future of Enzymology: An Appraisal....................... 521 39.1 Transition–State Analysis and Computational Enzymology.... 522 39.2 Single-Molecule Enzymology......................... 523 39.3 Structure-Function Dissection of Enzyme Catalysis.......... 524 39.4 Designing Novel Catalysts............................ 531 39.5 Enzymes Made to Order............................. 539 39.6 Summing Up..................................... 547 References............................................ 547 40 Closure – Whither Enzymology............................ 553 References............................................ 557 Bibliography.............................................. 559 About the Author N. S. Punekar is currently working as a Professor at prestigious Indian Institute of Technology (IIT) Bombay, Mumbai, India. He obtained his Ph.D. from the coveted Indian Institute of Sciences, Bangalore, India, in the year 1984 and subsequently worked as postdoctoral fellow at University of Wisconsin, Madison, USA, till 1988. He joined IIT Bombay as Assistant Professor in 1988 and subsequently got elevated to the rank of Professor in 2001. Dr. Punekar’s major research interest lies in microbial biochemistry and molecu- lar enzymology, microbial metabolic regulation, understanding metabolism through biochemical and recombinant DNA techniques, and fungal molecular genetics and its applications to metabolic engineering. Dr. Punekar has published around 50 papers in peer-reviewed reputed journals and more importantly has 4 patents to his credit. He has been an excellent teacher of Enzymology and Industrial Microbi- ology, which is evident by the fact that he has received the “Excellence in Teaching Award” at IIT Bombay in the years 2000, 2012 and 2018. He possesses more than two decades of experience of teaching Enzymology at IIT Bombay. He is associated with various societies and committees of Government of India as an expert member. He is also in editorial board of journals like Indian Journal of Biochemistry and Biophysics, Indian Journal of Biotechnology, Indian Journal of Experimental Biology, etc. xxiii Part I Enzyme Catalysis – A Perspective Enzymes: Their Place in Biology 1 One marvels at the intricate design of living systems, and we cannot but wonder how life originated on this planet. Whether ﬁrst biological structures emerged as the self- reproducing genetic templates (genetics-ﬁrst origin of life) or the metabolic univer- sality preceded the genome and eventually integrated it (metabolism-ﬁrst origin of life) is still a matter of hot scientiﬁc debate. There is growing acceptance that the RNA world came ﬁrst – as RNA molecules can perform both the functions of information storage and catalysis. Regardless of which view eventually gains accep- tance, emergence of catalytic phenomena is at the core of biology. The last century has seen an explosive growth in our understanding of biological systems. The progression has involved successive emphasis on taxonomy ! physiology ! bio- chemistry ! molecular biology ! genetic engineering and ﬁnally the large-scale study of genomes. The ﬁeld of molecular biology became largely synonymous with the study of DNA – the genetic material. Molecular biology however had its beginnings in the understanding of biomolecular structure and function. Apprecia- tion of proteins, catalytic phenomena, and the function of enzymes had a large role to play in the progress of modern biology. Enzymes and catalytic phenomena occupy a central position in biology. Life as we know it is not possible without enzyme catalysts. Greater than 99% of reactions relevant to biological systems are catalyzed by protein catalysts. A few RNA-catalyzed reactions along with all the uncatalyzed steps of metabolism occupy the rest 1%. While it may do to explain living beings as open systems that exchange matter and energy with their environment – thermodynamic feasibility alone is insufﬁcient to be living! Kinetic barriers have to be overcome. Reactions with relatively fast uncatalyzed rates, like removal of hydrogen peroxide or hydration of carbon dioxide, also need to be accelerated. Enzymes are thus a fundamental necessity for life to exist and progress. The key to knowledge of enzymes is the study of reaction velocities, not of equilibria. After all living beings are systems away from equilibrium. # Springer Nature Singapore Pte Ltd. 2018 3 N. S. Punekar, ENZYMES: Catalysis, Kinetics and Mechanisms, https://doi.org/10.1007/978-981-13-0785-0_1 4 1 Enzymes: Their Place in Biology Enzymology – the study of enzymes – has been an autocatalytic intellectual activity. Apart from knowledge gained on their structure and function, the study of enzymes is a driving force in advancing our understanding of biological phenomena as diverse as intermediary metabolism and physiology, molecular biology and genetics, cellular signaling and regulation, and differentiation and development. The conﬁdence in our experience with enzymes is so strong that they have found applications in a variety of industries including food, pharmaceuticals, textiles, and the environment. We encounter enzymes in every facet of biology and are forced to admire their exquisite roles. Enzymes were excellent models and earliest examples to understand protein structure-function. These include enzymes like hen egg white lysozyme, bovine pancreatic ribonuclease A (RNase A), trypsin, and chymotrypsin. A few of these were encountered during the study of digestive processes. Selectivity of proteases was exploited, and they served as useful reagents to cleave and study protein structure. The ﬁeld of molecular biology has beneﬁted enormously from enzymatic tools to cut, ligate, and replicate information molecules like DNA and RNA. Metabolic and cellular regulation is unthinkable without involving enzymes and their response to various environmental cues. The complexity associated with life processes owes it largely to their catalytic versatility, exquisite speciﬁcity, and ability to be modulated. Current advances in crystallography, electron microscopy, NMR, mass spectrom- etry, and genetic engineering have made it possible to view an enzyme closely while in action. Reverse genetics and genomics have made enzymology more powerful. Enzymology begins with a deﬁned function and its puriﬁcation; after which homing on to the corresponding gene has become very easy. Picomoles of pure enzyme protein are enough to determine its partial peptide sequence and obtain a ﬁngerprint. From here it is a well-beaten track of gene identiﬁcation, cloning, overexpression, and manipulation. Enzymes are superbly crafted catalysts of nature, and they are at the heart of every biological understanding. Life has literally preserved its past as chemistry. The book of life is written in the language of carbon chemistry, and enzymes form a major bridge between chemistry and biology. Enzymology is the domain where chemistry signiﬁcantly intersects biology and biology is at its quantitative best. From early history the evergreen tree of enzymology was nurtured by chemical and biological thought. We will take a look at this rich history in the next section. Suggested Reading Cleland WW (1979) Enzymology-dead? Trends Biochem Sci 4:47-48 Khosla C (2015) Quo vadis, enzymology? Nat Chem Biol 11:438–441 Kornberg A (1987) The two cultures: chemistry and biology. Biochemistry 26:6888–6891 Zalatan JG, Herschlag D (2009) The far reaches of enzymology. Nat Chem Biol 5:516–520 Enzymes: Historical Aspects 2 Historically, the ﬁeld of enzymology was born out of practical and theoretical considerations. A perusal of early enzyme literature indicates that the ﬁeld has evolved from fundamental questions about their function, their nature, and their biological role. This chapter outlines the course of historical development of enzy- mology, and some of these landmarks are listed in Table 2.1. 2.1 Biocatalysis: The Beginnings Past human industry like cheese making provided insights into some properties of enzymatic processes. The earliest recorded example of cheese making contains reference to extracts of ﬁg tree – a source of the proteolytic enzyme ﬁcin. Only later did rennet (a source of another protease chymosin) become popular in cheese processing. Meat tenderizing is the other application that implicitly used enzymes over the years. Apart from the ﬁg tree extract, the fruit and other parts of papaya (Carica papaya; contains the now well-known proteolytic enzyme papain) have found early utility in meat tenderizing. Indeed the work on gastric digestion of meat – proteases in particular – by Rene Reaumur (1751) and Lazzaro Spallanzani (1780) laid a scientiﬁc foundation for the study of enzyme catalysis. Reaumur’s experiments with digestion of meat represent the ﬁrst systematic record of the activity due to an enzyme. However the term enzyme was yet to be coined then! Theodor Schwann used the word pepsin in 1836 for the proteolytic activity of the gastric mucosa. He also conducted careful quantitative experiments, to establish that acid was necessary but not sufﬁcient for this reaction to take place. Among his many other contributions, Schwann also coined the term metabolism. Parallel to the work on proteolytic enzymes, developments with fermentation and on starch hydrolysis have equally contributed to the initial growth of enzymology. # Springer Nature Singapore Pte Ltd. 2018 5 N. S. Punekar, ENZYMES: Catalysis, Kinetics and Mechanisms, https://doi.org/10.1007/978-981-13-0785-0_2 6 2 Enzymes: Historical Aspects Table 2.1 Landmarks in enzyme studies (enzymology classics) Year (Discovery/ Author(s) Publication) Contribution R. Reaumur 1751 Gastric digestion in birds L. Spallanzani 1780 Digestion of meat by gastric juice A. Payen & J. Persoz 1833 Amylase (diastase) activity J. Berzelius 1836 Catalysis as a concept W. Kuhne 1867 “Enzyme” term deﬁned J. Takamine 1894 Patent on fungal diastase E. Fischer 1894 Lock and key concept G. Bertrand 1897 Co-ferment (coenzyme) conceived P.E. Duclaux 1898 Enzyme names to end with sufﬁx “ase” V. Henri 1903 Hyperbolic rate equation S.P.L. Sorensen 1909 pH scale and buffers L. Michaelis & M. Menten 1913 Equilibrium treatment for ES complex R.M. Willstatter 1922 Trager theory of enzyme action G.E. Briggs & J.B.S. Haldane 1925 Steady-state treatment for ES complex J.B. Sumner 1926 Urease – Puriﬁcation and crystallization H. Lineweaver & D. Burk 1934 Double reciprocal plot (1/v versus 1/[S]) K. Stern 1935 First ES complex observed M. Doudoroff 1947 Radioisotope use in enzyme mechanisms A.G. Ogston 1948 Asymmetric interaction with substrate L. Pauling 1948 Enzyme binds TS better than S F. Westheimer 1951 Enzymatic hydride transfer (2H, 3H used) D.E. Koshland Jr. 1958 Induced ﬁt hypothesis C.H.W. Hirs et al. 1960 First enzyme sequenced – RNase A Enzyme commission 1961 Enzyme classiﬁcation and nomenclature D.C. Phillips et al. 1962 First enzyme structure – lysozyme W.W. Cleland 1963 Systematization of enzyme kinetic study J. Monod et al. 1965 Model for allosteric transitions R.B. Merriﬁeld 1969 Chemical synthesis of RNase A S. Altman & T.R. Cech 1981 Catalysis by RNA molecules Gottlieb Kirchhoff discovered plant amylase (later identiﬁed as α-amylase) activ- ity while characterizing the hydrolysis of starch to sugar. He demonstrated the acid- facilitated conversion of starch to sugar and clearly recognized that the formation of sugar from starch during germination of grain is akin to chemical hydrolysis (1815). The work of Kirchhoff on starch hydrolysis was extended by Anselme Payen and Jean Persoz (1833). They enriched (ﬁrst attempts of enzyme puriﬁcation!) the hydrolytic activity from malt gluten and termed it as diastase. The name diastase (Greek; diastasis – to make a breach) has signiﬁcantly inﬂuenced the development of the ﬁeld of enzymology since then (see below). Yet another source of a starch hydrolyzing activity was identiﬁed in saliva by Erhard Leuchs (1931). Remarkably, this report also invoked the possible practical utility of this activity. 2.2 “Enzyme”: Conceptual Origin 7 The two non-hydrolytic enzyme activities reported early include the peroxidase activity from horseradish and catalase. These two enzymes were recognized much ahead of the study of oxidative enzymes in early twentieth century. Work on catalase by Louis Thenard (1819) is the ﬁrst quantitative study of an enzymatic reaction. He also anticipated that such activities may be found in other animal and vegetable secretions. Enzymology ﬁnds its roots in some of the greatest names since eighteenth century, both in chemistry and biology. Clearly this subject is a true and sturdy bridge between contemporary chemistry and biology. Among the greats who contributed to its early development include Reaumur, Spallanzani, Thenard, Schwann, Berzelius, Liebig, Berthelot, Pasteur, Buchner, and Fischer. Many funda- mental contributions were made to enzymology by chemists of fame like Berzelius, Liebig, and Berthelot. It is however important to note that historically, the idea of catalysis arose because of the study of enzymes and their action. Jons Jacob Berzelius was the ﬁrst to deﬁne the term “catalyst” in 1836. In his view, a catalyst was a substance capable of wakening energies dormant, merely by its presence. He was also the ﬁrst to recognize the similarity of catalysis in a chemical reaction and inside a living cell. However, Berzelius made no distinction between the catalytic phenomenon occurring in animate and inanimate world. He also used the now famous words isomer, polymer, ammonium, protein, and globulin. Those were the times when a “vital force” was associated with living cells and biocatalysts were part of this explanation. Only much later did the concept take root that ordinary physical and chemical principles apply to enzyme catalysis. Pierre Berthelot was the ﬁrst to derive a second-order rate equation which inﬂuenced the publication by Guldberg and Waage on law of mass action leading to chemical kinetics. As a part of their study on catalytic phenomena, Wohler and Liebig discovered “emulsin” (a β-glucosidase) from almonds in 1837. Indeed this enzyme was cleverly used by Fischer subsequently (almost 50 years later!) to deﬁne enzyme speciﬁcity. 2.2 “Enzyme”: Conceptual Origin Swedish chemist Berzelius (1779–1848) proposed the name catalysis (from the Greek kata, wholly, and lyein, to loosen) in 1836. When Berzelius ﬁrst invoked the term “catalysis,” he did not make any distinction between the chemical catalysis and catalysis in (or by) biological systems. He used a generic term “contact sub- stance” for a catalyst. The origin of the word “enzyme” dedicated to biological catalysts has a convoluted history. Much of this drama was played out during a vigorous debate on whether there is special force (the “vital force”) associated with reactions occurring in living systems. Ever since Payen and Persoz (1833) introduced this name for the starch hydrolytic activity, “diastase” has often been used to generally mean a catalyst of biological origin. In fact Victor Henri in his 8 2 Enzymes: Historical Aspects 1903 book on enzyme kinetics (an early classic on enzyme action) used diastase to mean an “enzyme.” Many other French scientists including Pierre Duclaux and Gabriel Bertrand did use diastase to mean what we now call enzymes. The sufﬁx “ase” – arising out of diastase – was subsequently recommended for all enzyme names (by Duclaux in 1898). Ferment as a term was used to describe both living yeast and the action of its cellular contents. Berthelot’s extraction of “ferment” (1860) from yeast cells marks the beginning of action of enzymes outside of a living cell. This also dealt a blow to vitalistic thinking in biochemistry. The analogy between ferment-catalyzed and acid- catalyzed hydrolysis of starch was well-recognized by the successive contributions of Kirchhoff, Payen and Persoz, and Berzelius. Schwann had used a similar analogy for pepsin. Willy Kuhne in 1867 extended this further to pancreatic digestion of proteins and called this activity trypsin in 1877. The essential meaning of “ferment” was consolidated by Kuhne; subsequently the word enzyme (in yeast) was ﬁrst used by him in 1877. In fact trypsin was the ﬁrst candidate “ferment” to be called an enzyme. The evolution and acceptance of word enzyme have taken its time. Both the descriptions – “diastase” (mostly in French scientiﬁc literature) and “ferment” – were used occasionally well into the early twentieth century. The vitalistic theory was ﬁrmly laid to rest with Eduard Buchner’s conclusive demonstration that suitable extract from yeast cells could convert sucrose to alcohol. This was revolutionary in 1897 since fermentation was shown to occur “without living yeast” for the ﬁrst time. The activity was ascribed to a single substance which was named “zymase” (and alcoholase by Emile Roux). It is now history that this activity in fact represents the entire glycolytic sequence of reactions. Out of contro- versy on the nature of alcoholic fermentation, the word “enzyme” was born. This word reminds us that yeast (“zyme”) and its activities were resolved through the prisms of biology and chemistry to create the rich domain of enzymology. 2.3 Key Developments in Enzymology Protein Nature of Enzymes Early progress on enzymes was impeded because not much was known on the chemical nature of proteins. Much less was known about the chemical nature of enzymes. One approach to understand them was to purify them for detailed analysis. Kuhne and Chittenden extensively used the technique of protein fractionation by ammonium sulfate and also introduced the use of dialysis and dialysis tubing (1883). Powerful methods to purify enzymes were developed by Richard Willstatter – the ﬁrst introduction of alumina Cγ gel was made. Peroxidase was taken to such high level of purity that the preparation failed in then prevailing tests for protein. This unfortunately led him to wrongly conclude that enzymes are not proteins (1926). The seminal discovery by James Sumner, proving that urease is a protein, therefore assumes great signiﬁcance (Sumner 1933). This view was further conﬁrmed by puriﬁcation and crystallization of three more enzymes – pepsin, trypsin, and chymotrypsin – by Northrop and Kunitz (between 1930 and 1935). It 2.3 Key Developments in Enzymology 9 must impress anyone to note that all this was accomplished by just two simple puriﬁcation techniques – fractional precipitation of proteins by ammonium sulfate and pH changes. Laccase is one of the early examples of an enzyme that was not a hydrolase. Bertrand (1895) described it as an “oxidase” and suggested that this enzyme contained a divalent metal. His description of “co-ferment” – a nonprotein compo- nent of laccase – is the ﬁrst descriptor of an enzyme cofactor. More powerful yet gentler procedures of protein puriﬁcation (and dialysis, etc.) hastened the progress of enzymology by providing many pure enzyme preparations. The end of the nineteenth century saw an increase in the number of reports on enzymes. By 1955 the number of enzymes reported was so large that their proper organization into categories became necessary. Under the auspices of the Interna- tional Union of Biochemists, the International Commission on Enzymes was established to systematize the classiﬁcation and naming of enzymes. As a result, the Enzyme Commission produced guidelines on enzyme nomenclature and brought out its recommendations in 1961. Kinetic Foundations Because they are excellent catalysts, enzyme kinetic behavior could be studied regardless of meager knowledge of their composition. Even after their protein nature was established, it has taken long to relate structural basis of enzyme kinetic behavior. As early as 1898, the reversibility of an enzyme reaction was reported. The enzymatic synthesis of a glucoside (maltose from glucose) by the yeast maltase established some key features: (a) an enzyme being a catalyst speeds up the reaction in both directions of a reversible reaction, (b) at least some steps in metabolism may go in either direction, and (c) enzymes may be involved in the cellular biosynthetic processes. The reversibility of enzyme catalysis brought it within the ambit of thermody- namic analysis and physical chemistry. The thermodynamic constraints imposed upon catalyzed and uncatalyzed reactions were set forth by J van’t Hoff. This subsequently led JBS Haldane to relate enzyme kinetic parameters with reaction thermodynamics and arrive at the famous Haldane relationship (Enzymes 1930). Yeast invertase has the singular distinction as the working example for early work on enzyme reaction kinetics and thermodynamics. AJ Brown (1902) deduced the formation of invertase-sucrose complex (the ES complex) from initial rate measurements. It was in 1903 that V. Henri for the ﬁrst time derived the hyperbolic rate equation for a single-substrate enzymatic reaction. He provided the general process used to derive such rate equation – an exercise central to any enzyme kinetic study. Henri also recognized that ‘the validity of a rate equation is necessary but not sufﬁcient to prove the postulated kinetic mechanism’. In fact the now famous Michaelis–Menten equation, based on the equilibrium treatment of the system, was published about 10 years later in 1913. A more general form of the Henri- Michaelis-Menten equation to describe enzyme kinetics was derived by Briggs and Haldane via the steady-state approach in 1925. We continue to use this fundamental equation even today to describe the substrate saturation phenomenon of an 10 2 Enzymes: Historical Aspects enzyme reaction. A very popular linear form of this hyperbolic relation between initial velocity and substrate concentration is attributed to Lineweaver and Burk (1934). A systematic study of enzyme reaction rates dictated that buffers be used to control hydrogen ion concentrations. This was indeed the impetus to the work published in 1909 by Sorenson on the pH scale and buffers. Subsequently Leonor Michaelis and others emphasized the importance of pH on enzyme activity and routinely controlled it in all their studies. The ES complex formation was a kinetic concept to begin with. First direct observation of an enzyme substrate complex of catalase was made by KG Stern (1935); he monitored the catalase–HOOEt complex using spectroscopy. Mechanistic Studies Emil Fischer was an unusual organic chemist of highest caliber. He was responsible for establishing the rigor of synthetic and analytical skills of organic chemistry to biological problems. As early as 1894, he observed that substrates for invertin (now the invertase or sucrose hydrolase) are not substrates for emulsin (a β-glucosidase) and vice versa. Fischer opined that “enzymes are fussy about the conﬁguration of their object of attack.” For example, the enzyme and the glucoside on which it acts must ﬁt each other like a “lock and key” to be able to catalyze the chemical reaction. The future, as we know it, conﬁrmed the genius of Fischer. This laid the foundation for describing fundamental properties of enzyme like speciﬁcity, stereoselectivity, and the famous lock-and-key analogy for enzyme– substrate interactions. In an attempt to explain how enzymes work, the “Trager” or carrier theory was proposed by Willstatter (in 1922). According to him enzymes contained smaller reactive groups that have afﬁnity toward speciﬁc groups on the substrate – leading to enzyme speciﬁcity. Of course, these reactive groups were thought to be attached to an inert colloidal carrier to form the enzyme. Clearly the fact that enzymes are proteins was not yet established then. The hypothesis by AG Ogston (1948) attempted to explain how enzymes achieve chemical asymmetry through three point contact with their substrates. This paved the way for further experiments in elucidating enzyme chemical mechanisms. Redox reactions involving pyridine nucleotides and the mechanism of hydride transfer followed shortly thereafter. Frank Westheimer and his colleagues, working with alcohol dehydrogenase and lactate dehydrogenase as examples, showed that the substrate hydrogen was transferred selectively to one side of the nicotinamide ring. This pioneering research in 1953 made use of deuterium- and tritium-labeled substrates to establish the stereospeciﬁcity of these hydride transfers. Work by Michael Duodoroff’s group (1947) on bacterial disaccharide phosphorylases forms an early and brilliant example of use of radioisotopes (32P phosphate) in the study of enzyme mechanisms. Two similar reactions involving disaccharide phosphorolysis, namely, sucrose phosphorylase and maltose phosphor- ylase, were shown to follow completely different mechanisms. This led directly to the notion of single displacement versus double displacement reactions and subse- quently the SN1 and SN2 reaction pathways. 2.3 Key Developments in Enzymology 11 The theory of kinetic criteria to distinguish enzyme mechanisms was elaborated by WW Cleland in three seminal papers (1963). At the least, this provided a common language to present enzyme kinetic data, for an otherwise confusing variety of notations found in enzyme kinetic literature. The impact of systematizing enzyme kinetics served two useful purposes – (1) it provided a common kinetic notation for presentation and (2) provided a summary of criteria on how to relate kinetic data with reaction mechanisms. The rigidity of enzyme active site structure became untenable over time. Com- plementarity of enzyme active site to accommodate the transition state structure (rather than the substrate or the product) by Linus Pauling (1946) was prophetic; this clearly anticipated the need for protein motion, however subtle, at enzyme active sites. The idea of conformational ﬂexibility of protein molecules as a prerequisite for enzyme activity superseded the earlier lock-and-key concept. This theme further matured into the concept of induced ﬁt hypothesis as proposed by Koshland (1958). The conformational ﬂexibility of a protein and ligand binding through induced ﬁt later became key elements of allosteric transitions. The plasticity of protein structure for regulation thus became inescapable (the famous Monod-Wyman-Changeux model to explain cooperative interactions in oligomeric proteins). Recognition that enzymes bring about enormous rate accelerations quickly led to a search for underlying principles of such catalysis. Attempts to demystify and explain enzyme catalysis in physicochemical terms were made. Different contrib- utory factors were dissected out through model chemical reactions as well as enzymes. The work of TC Bruice, WP Jencks, ML Bender, DE Koshland Jr., and others is signiﬁcant in this query. A combination of factors – intermolecular/ conformational effects, general acid/base catalysis, nucleophilic/electrophilic catalysis, etc. – contributed to accomplish remarkable rate accelerations observed with enzymes. It is now well-recognized that a combination of many factors produces an enzyme. However search for novel catalytic tools evolved by nature continues unabated even today. Structure and Synthesis The stamp of chemist’s contribution to the study of enzymes is obvious from the progression – isolation and structure elucidation followed by total synthesis. Insulin was the ﬁrst protein whose complete chemical structure was determined. However, among enzymes, this credit goes to bovine pancreatic ribonuclease A (RNase A) – it was the ﬁrst enzyme whose primary sequence was elucidated. But lysozyme (this was followed later by RNase A) is the ﬁrst enzyme whose three-dimensional structure was made available through X-ray crystallography. In a typical organic chemist’s approach, total synthesis of a molecule completes the structure elucidation process. In this sense, RNase A was the ﬁrst enzyme whose total synthesis was achieved (RB Merriﬁeld), and it culminated in a catalytically active protein. In summary, the history of enzymology is a rich source of factual and conceptual discoveries. Developments in this ﬁeld were accelerated by chemists and biologists in equal measure. Once established, enzymology revolutionized both the parent 12 2 Enzymes: Historical Aspects disciplines – biology and chemistry. This is amply evident from the list of Nobel laureates (Table 2.2) and their work recognized by the two scientiﬁc communities that nurtured the study of enzymes. Table 2.2 Nobel laureates who contributed to the growth of enzymology Scientist Yeara Enzymology – topic of study E. Fischer 1902-C Stereochemistry and lock-and-key concept S. Arrhenius 1903-C Activation energy and catalysis E. Buchner 1907-C Cell-free extracts and fermentation A. Harden and H. von Euler 1929-C Coenzymes and fermentation C. Eijkman and F.G. Hopkins 1929-M Vitamins, nutrition, and coenzymes O. Warburg 1931-M Respiratory enzymes A. Szent-Gyorgyi 1937-M Fumarate catalysis of TCA cycle R. Kuhn 1938-C Vitamins and coenzymes A. Fleming 1945-M Penicillin and lysozyme J.B. Sumner, J.H. Northrop, 1946-C Puriﬁcation and crystallization of enzymes and M. Kunitz C. Cori and G. Cori 1947-M Enzymes of glycogen metabolism H.A. Krebs and F. Lipmann 1953-M TCA cycle and coenzyme A L. Pauling 1954-C Secondary structure – α helix; concept that enzyme binds the transition state H. Theorell 1955-M Oxidative enzyme mechanisms A.R. Todd 1957-C Nucleotides and nucleotide coenzymes F. Sanger 1958-C Insulin sequence through proteases S. Ochoa and A. Kornberg 1959-M Nucleic acid biosynthesis enzymes M.F. Perutz and 1962-C Crystal structure of globular proteins J.H. Kendrew D. Crowfoot Hodgkin 1964-C Structure of vitamin B12 K. Bloch and F. Lynen 1964-M Cholesterol and fatty acid enzymes F. Jacob, A. Lwoff, and 1965-M Genetic control of enzyme synthesis and allostery J. Monod L.F. Leloir 1970-C Sugar nucleotides and carbohydrate biosynthesis E.W. Sutherland Jr. 1971-M Enzyme and metabolic regulation by cAMP C.B. Anﬁnsen, S. Moore, and 1972-C Chemical structure – Catalytic activity of RNase A W.H. Stein J.W. Cornforth 1975-C Stereochemistry of enzyme reactions W. Arber, D. Nathans, and 1978-M Restriction endonucleases H.O. Smith F. Sanger 1980-C DNA sequencing (ddNTP method) with enzymes R.B. Merriﬁeld 1984-C Chemical synthesis of RNase A J.W. Black, G.B. Elion, and 1988-M Inhibitors (enzyme) as drugs G.H. Hitchings S. Altman and T.R. Cech 1989-C Catalysis by RNA molecules E.H. Fischer and E.G. Krebs 1992-M Protein kinases and protein phosphorylation K.B. Mullis 1993-C Polymerase chain reaction (continued) Reference 13 Table 2.2 (continued) Scientist Yeara Enzymology – topic of study P.D. Boyer, J.E. Walker, and 1997-C ATP synthase and Na/K-ATPase J.C. Skou A.H. Zewail 1999-C Detection/existence of transition state I. Rose (and others) 2004-C Ubiquitin-protein degradation and isotope exchanges in enzymology A. Warshel (and others) 2013-C Computational enzymology F.H. Arnold 2018-C Directed evolution of enzymes a Prize awarded this year for C chemistry, M physiology and medicine Reference Sumner JB (1933) The chemical nature of enzymes. Science 78:335 Suggested Reading Bugg TDH (2001) The development of mechanistic enzymology in the 20th century. Nat Prod Rep 18:465–493 Cornish-Bowden A (1999) The origins of enzymology. Biochemist 19:36–38 Cornish-Bowden A (2013) The origins of enzyme kinetics. FEBS Lett 587:2725–2730 Friedmann HC (1981) Enzymes: benchmark papers in biochemistry. Hutchinson Ross Publishing Company, Stroudsburg Johnson KA (2013) A century of enzyme kinetic analysis, 1913 to 2013. FEBS Lett 587:2753–2766 Neidleman SL (1990) The archeology of enzymology. In: Abramowicz DA (ed) Biocatalysis. Van Nostrand Reinhold, New York, pp 1–24 Exploiting Enzymes: Technology and Applications 3 Much before enzymes were identiﬁed as discrete biochemical entities, they found favor through their useful properties. Early applications included use of enzyme preparations in meat tenderizing and starch hydrolysis. From the very beginning, commercial enzyme applications have largely belonged to a group of hydrolytic reactions. But a few oxidative enzymes were also exploited. While this trend holds even today, examples of designer enzymes and catalysts for more complex chemical processes are being developed. The ﬁrst application of diastase (α-amylase) was by Jokichi Takamine. His 1894 patent (US Patent No. 525823) describes a process to make Taka-diastase from Aspergillus oryzae. This α-amylase was useful as a digestive aid, in eliminating starchy material from textiles and laundry. In a short but succinct paper, E.F. Leuchs (in 1931) described “the action of saliva on starch.” The possible practical utility of such activity was clearly anticipated by him. The last line of his report reads “it will be possible to use saliva and gastric juice of killed animals very successfully in cases of defective digestion.” The quantity and quality of an enzyme are two critical parameters that deﬁne their application and extent of use. Industrial scale processes require enzymes (often in crude form) in tons, whereas precise clinical use mandates extreme purity and minimal or no contaminating factors. Accordingly, the enzyme production costs for different end objectives vary – they can be of high volume and low cost or low volume but of high cost. For instance, medically valuable products like streptokinase and asparaginase need to be very pure and are therefore expensive. Enzyme catalysts of practical import are sought by industries in many different ways. Signiﬁcant among these are screening for useful activities from the naturally abundant diversity, modifying already available enzyme properties to suit our requirements, and geneti- cally engineering desirable properties into these catalysts. We will brieﬂy touch upon the applications of enzymes and industrial strategies with suitable examples in this chapter. Applications of enzymes and enzyme technology can occupy volumes, and many authoritative books are available for the interested reader. # Springer Nature Singapore Pte Ltd. 2018 15 N. S. Punekar, ENZYMES: Catalysis, Kinetics and Mechanisms, https://doi.org/10.1007/978-981-13-0785-0_3 16 3 Exploiting Enzymes: Technology and Applications 3.1 Exploiting Natural Diversity The rich biodiversity on earth goes hand in hand with naturally vast array of catalytic activities. A cleverly designed screen almost always leads to an enzyme with desired properties. Thermostable protease (from Bacillus strains) and DNA polymerase (from Thermus aquaticus) are two examples of enzymes chosen for high temperature stability. The range of natural diversity is obvious from the number of enzymes that have found niche applications in the processing of carbohydrate polymers, proteins, and lipids. Enzymes for Bioprocessing Polysaccharides are the major biomolecules that com- prise biomass on this planet. They serve two important functions – energy storage (such as starch) and structural rigidity (such as cellulose). It is therefore not surprising that enzyme technology took its roots through processes to hydrolyze these sugar polymers. Microbes (bacteria and fungi) constitute an abundant source of amylases and cellulases. Controlled hydrolysis of starch to sweeteners (and sugar substitutes) is a well-developed industry (Fig. 3.1). Various enzymes used in the starch sacchariﬁcation process are α-amylases, β-amylases, glucoamylases, pullulanases, and glucose isomerase. Despite certain limitations, conversion of glucose to fructose through glucose isomerase is central to many sucrose substitutes – with distinct economic and manufacturing advantages. Although there is an abundance of cellulose in nature, transforming cellulosic biomass into sugar has been a challenge. Concerted action of a bunch of enzymes (that constitute the “cellulase complex”) is required for this (Payne et al. 2015). Signiﬁcant advances in enzymatic processes to breakdown cellulose into ferment- able sugars are being made. In the meanwhile, individual components (Table 3.1) of the cellulase complex have found application in textile and paper industry. Proteases and lipases are next in order of signiﬁcance in enzyme industry. Apart from the historical signiﬁcance of papain and digestive enzymes (like trypsin and chymotrypsin), this class of enzymes has found wide-ranging applications in foods, detergents, and tanning of leather. Bacteria and fungi are ideal sources for the large- scale production of proteases (Li et al. 2013). Most important alkaline protease producers are Bacillus strains and fungi belonging to genus Aspergillus. Subtilisin is the best known bacterial protease additive of modern detergents. It has been exten- sively selected/modiﬁed for features like pH optimum and temperature stability. Chymosin (also known as rennin) is a milk coagulating enzyme from calf stomach, which is used for generations in cheese making. An equivalent enzyme from a microbial source was sought, and several Mucor strains are chosen to produce rennin substitutes. Many other enzymes including lipases and pectinases are also available in industrial scale. A representative list of enzymes commonly used in industry is given in the table (Table 3.2). 3.1 Exploiting Natural Diversity 17 Cyclodextrins Cyclomaltodextrin-D -glucotransferase a–D-Glucosidase a–Amylase STARCH Pullulanase b–Amylase Glucoamylase Glucose isomerase Glucose Fructose Fig. 3.1 Signiﬁcant steps and enzymes employed in starch processing. Glucose residues of starch are schematically represented as circles. Filled circles indicate glucose residues whose C1-OH has not entered into a glycosidic linkage (free reducing ends). Besides glucose isomerase, all the possible enzymatic modes of dismantling starch are shown. Some combinations of these enzymes are commercially available as industrial formulation Enzymes in Pharma and Medical Applications Pharmaceutical industry is another big beneﬁciary of applied enzymology. Enzymes isolated from natural sources as well as those cloned and expressed (through genetic engineering) are in use. Enzymes and their critical study serve multiple purposes in drug discovery and development. Active principles of many effective drugs are enzyme inhibitors (Robertson 2005). An enzyme, critically located in the intermediary metabolism, may provide an excellent target to screen for such inhibitors. A few successful examples of drugs have panned out from such enzyme screens. 18 3 Exploiting Enzymes: Technology and Applications Table 3.1 Component activities of cellulase complex and their applications Cellulase component Substrate speciﬁcity Application β-Glucosidase Cellobiose!Glucose Sacchariﬁcation (Cellobiase) Cellobiohydrolase I Cellulose! Cellobiose (exo – Biomass conversion (CBH1) Nonreducing end) Cellobiohydrolase II Cellulose! Cellobiose(both exo and endo) Biomass conversion (CBH2) Endoglucanase I (EG1) Cellulose (endo) Textile/fabric softening, Biopolishing Endoglucanase II (EG2) Cellulose (endo) Textile/fabric softening, Biopolishing Xylanase Xylan Paper pulp deinking All components Cellulose and Xylan Feed/fodder, biomass Conversion Table 3.2 Large-scale use of enzymes in industry Enzyme Application Acting on carbohydrates Amylases Starch processing Cellulase complex Biomass conversion, textile industry Pectinases, esterases Food industry, fruit juice, brewing Glucose isomerase, invertase High fructose syrups, invert sugar Acting on proteins Papain, pepsin Meat and leather processing, treating dough Rennin, chymosin Cheese making Subtilisin Detergents, leather and wool processing Acting on lipids and esters Lipases Food and detergent industry, cocoa butter Acting on antibiotics Penicillin acylase Produce 6-aminopenicillanic acid (6-APA) The concept of screening for enzyme inhibitors was ﬁrst adopted by Hamao Umezawa’s group in Japan (Umezawa 1982). Since then, many enzyme inhibitors have been discovered (few are listed in Table 3.3) and are in use. For instance, preventing absorption of dietary fat (triglycerides) can be a possible strategy to control obesity. An appropriate lipase from the digestive juices could serve as a target for this screening (Fig. 3.2). Often the active chemical entity obtained from an enzyme screen may not ﬁnd direct application. These lead compounds (inhibitors) are suitably altered/derivatized 3.1 Exploiting Natural Diversity 19 Table 3.3 Examples of enzyme-targeted screens for active principles Enzyme target Screening outcome End use Pepsin Pepstatin Ulcers Angiotensin converting enzyme Captopril Hypertension HMG CoA reductase Lovastatin Hypercholesteremia α-Amylase Acarbose Diabetes Triacylglycerol lipase Orlistat (lipostatin) Obesity Acetylcholine esterase Rivastigmine Alzheimer’s disease β-Lactamase Clavulanic acid Combination therapy Fig. 3.2 Flow chart Digestion of FAT outlining the design of a lipase inhibitor screen Lipase (involved in hydrolysis of triacylglycerols) Inhibition (lipase activity - enzyme assay) Inhibitor Screen (natural or synthetic chemical libraries) Lipostatin (active principle) to achieve better bioavailability and reduce toxicity. An in-depth kinetic analysis of enzyme inhibition (concluded in Chap. 28) is at the heart of modern drug discovery programs. Besides being targets for inhibitor screens, many enzymes are employed as catalysts for synthesis. The whole range of β-lactam antibiotics available today includes a large number of semisynthetic penicillins and cephalosporins. While penicillin G is produced by fermentation, 6-aminopenicillanic acid – an important precursor for semisynthetic penicillins – is derived from it (Fig. 3.3). Penicillin acylase is a valuable commodity in the large-scale production of 6-aminopenicillanic acid. Because of their catalytic potential coupled with speciﬁcity, many enzymes are used as exquisite analytical tools. Alkaline phosphatase and peroxidase are two reporter enzymes of extensive history in ELISA (enzyme-linked immunosorbent assay). Here the desired speciﬁcity of interaction (through antibodies) is coupled to the signal ampliﬁcation provided by enzyme catalysis. Enzymes as antibody- conjugates ﬁnd routine use in detection of DNA/RNA/protein on blots. Taq DNA polymerase is extensively employed for DNA ampliﬁcation through polymerase chain reaction (PCR). A number of metabolites are analyzed (in a clinical setting) 20 3 Exploiting Enzymes: Technology and Applications O H N S O N O H O O Penicilloic acid O H b–Lactamase N S N O O Penicillin G O Penicillin acylase O OH H 2N S + Phenylacetic acid N O O O 6-Aminopenicillanic acid O H N R S Semisynthetic N O Penicillins O O O O Cephalosporins Fig. 3.3 Enzymes and steps relevant to penicillin (β-lactams) industry. Antibiotic resistance is often due to a β-lactamase; better antibiotics may be evolved by screening for novel structures that are not acted upon by the β-lactamase. Penicillin acylase is used to produce 6-aminopenicillanic acid (6-APA). 6-APA is an important precursor to make semisynthetic penicillins, both through enzymatic and chemical routes through assays involving enzymes. A list of more commonly used enzymes and the corresponding analytes are given in Table 3.4. Some of these enzymes are also employed as a component of biosensors (see below). Enzymes have found medical applications in terms of diagnosis as well as therapy. A few enzymes ﬁnd direct application as therapeutic agents in medicine. Best known examples from the market include diastase (α-amylase, digestive aid), asparaginase (leukemia, antitumor therapy), rhodanese (cyanide poisoning), and streptokinase (medication to dissolve blood clots). However, a large number 3.1 Exploiting Natural Diversity 21 Table 3.4 Examples of Enzyme Analyte detected/estimated enzymes for metabolite Catalase Hydrogen peroxide analysis Glucose oxidase Glucose Hexokinase Glucose Alcohol dehydrogenase Ethanol Lactate dehydrogenase Lactate/pyruvate Luciferase ATP Urease Urea Cholesterol oxidase Cholesterol Table 3.5 Examples of enzymes as clinical markers Enzyme Used as marker for Lactate dehydrogenase (H4 isoform) Heart diseases Glutamate-oxoglutarate transaminase (SGOT), glutamate- Liver function pyruvate transaminase (SGPT) Creatine kinase Myocardial infarction, skeletal muscle damage Lactase Lactose intolerance Hexosaminidase A Tay-Sachs disease Acid phosphatase Prostate cancer Phenylalanine hydroxylase Phenylketonuria (PKU) of enzymes are routinely monitored as clinical markers (Table 3.5). Enzyme proﬁles from serum, amniotic ﬂuid, urine, etc. are monitored, and their levels are often correlated with disease conditions and aid in the diagnosis of disease. Enzymes and Issues of Safety Industrial preparation and use of enzymes come with its own safety and regulatory issues. Potential hazards from exposure to large quantities of a given enzyme include allergenicity, functional toxicity, chemical toxicity, and source-related contaminants. Large-scale manufacture of enzymes often ends with partially pure yet enriched preparations. Such material could contain potentially toxic chemicals (like mycotoxins) carried over from the source. Not all microorganisms are safe, and their trace contamination in the ﬁnal enzyme preparations requires attention. For enzymes to be used in food ingredients, they must be GRAS (generally recognized as safe). Some enzymes like proteases are potentially dangerous – particularly upon exposure of sensitive tissues to concentrated preparations. Since enzymes are proteins, they can be potent allergens. Repeated exposure through inhalation or skin contact can trigger severe allergic response. Enzyme preparations especially handled in the form of dust, dry powder, or aerosol are harmful and must be avoided. Many issues of safety regarding free enzyme preparations may be overcome by using them in the immobilized, granulated, or encapsulated form. 22 3 Exploiting Enzymes: Technology and Applications 3.2 Modifying Enzymes to Suit Requirements Despite the vast natural diversity of biological catalysts, signiﬁcant technology has developed to alter the properties of available enzymes. This tinkering has involved facets of their immobilization, chemical modiﬁcation, genetic engineering, or their use in nonaqueous solvents. Immobilization for Better Use Most natural enzymes isolated are in water soluble state. They cannot be stored in this form for long, often due to instability. Their immobilization is one way to enhance their shelf life (Mateo et al. 2007). In addition, immobilized enzymes are easy to recover and amenable to repeated use. This is an important consideration when the cost of enzyme is very high. The characteristics of the matrix are very critical in determining the performance of the immobilized enzyme system. These supports may be inorganic or organic according to the nature of their chemical composition. The physical characteristics like mean particle size, swelling behavior, mechanical strength, etc. decide the technical conditions in which the system is used. Enzymes may be quarantined on the matrix either irreversibly (by covalent bonding, entrapment, microencapsulation, cross-linking, etc.) or reversibly (by adsorption, ionic binding, afﬁnity binding, disulﬁde bonds, or che- late/metal binding). The cost associated with the process of immobilization determines whether it is economically viable to do so. Different means are adopted for enzyme immobilization in practice, and only a few are represented in Fig. 3.4. The ﬁeld of enzyme immobilization technology and its applications has grown vastly over the years. Many books and volumes (Methods in Enzymology series) are available for detailed reference (Brena and Batista-Viera 2006). This section covers very brieﬂy on this applied aspect of enzymes, and the reader is encouraged to refer the more specialized literature for the purpose. The choice of immobilization method depends on the type of the enzyme and the nature of applications in question. Non-covalent conﬁnement (like physical entrap- ment, microencapsulation, or electrostatic adsorption) methods at times may lead to enzyme leaching during operation. Covalent anchoring of enzymes on the other hand requires bifunctional cross-linking reagents and suitable functional groups on the enzyme surface. These functional groups must not be critical for enzyme activity however. A great deal of sophisticated chemistry has been developed to activate inert organic/inorganic polymers for subsequent enzyme immobilization. Carrier-bound (covalently linked to polymers; ﬁgure above) penicillin acylase is highly effective in the preparation of 6-APA and permits economic recycling of the catalyst. Penicillin acylase immobilized cassettes are available that function at >99% conversion efﬁ- ciency even after 1500 cycles of use. Glutaraldehyde is used to cross-link glucose isomerase – the cross-linked material can be reused many times

ENZYMES: Catalysis, Kinetics and Mechanisms 2018 PDF

Document Details

Tags

Related

Summary

Full Transcript

Upgrade to continue