Enzymes (Second Edition)
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ACKNOWLEDGMENTS
It is a great pleasure for me to thank the many friends and coworkers who have helped me in the preparation of this work. Many of the original lecture notes from which this text has developed were generated while I was teaching a course on biochemistry for first-year medical students at the University of Chicago, along with the late Howard S. Tager. Howard contributed greatly to my development as a teacher and writer. His untimely death was a great loss to many of us in the biomedical community; I dearly miss his guidance and friendship.
As described in the Preface, the notes on which this text is based were significantly expanded and reorganized to develop a course of enzymology for employees and students at the DuPont Merck Pharmaceutical Company. I am grateful for the many discussions with students during this course, which helped to refine the final presentation. I especially thank Diana Blessington for the original suggestion of a course of this nature. That a graduate-level course of this type could be presented within the structure of a for-profit pharmaceutical company speaks volumes for the insight and progressiveness of the management of DuPont Merck. I particularly thank James M. Trzaskos, Robert C. Newton, Ronald L. Magolda, and Pieter B. Timmermans for not only tolerating, but embracing this endeavor.
Many colleagues and coworkers contributed suggestions and artwork for this text. I thank June Davis, Petra Marchand, Diane Lombardo, Robert Lombardo, John Giannaras, Jean Williams, Randi Dowling, Drew Van Dyk, Rob Bruckner, Bill Pitts, Carl Decicco, Pieter Stouten, Jim Meek, Bill DeGrado, Steve Betz, Hank George, Jim Wells, and Charles Craik for their contributions.
Finally, and most importantly, I wish to thank my wife, Nancy, and our children, Lindsey and Amanda, for their constant love, support, and encouragement, without which this work could not have been completed.
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PREFACE TO THE FIRST EDITION
The latter half of this century has seen an unprecedented expansion in our knowledge and use of enzymes in a broad range of basic research and industrial applications. Enzymes are the catalytic cornerstones of metabolism, and as such are the focus of intense research within the biomedical community. Indeed enzymes remain the most common targets for therapeutic intervention within the pharmaceutical industry. Since ancient times enzymes also have played central roles in many manufacturing processes, such as in the production of wine, cheese, and breads. During the 1970s and 1980s much of the focus of the biochemical community shifted to the cloning and expression of proteins through the methods of molecular biology. Recently, some attention has shifted back to physicochemical characterization of these proteins, and their interactions with other macromolecules and small molecular weight ligands (e.g., substrates, activators, and inhibitors). Hence, there has been a resurgence of interest in the study of enzyme structures, kinetics, and mechanisms of catalysis.
The availability of up-to-date, introductory-level textbooks, however, has not kept up with the growing demand. I first became aware of this void while teaching introductory courses at the medical and graduate student level at the University of Chicago. I found that there were a number of excellent advanced texts that covered different aspects of enzymology with heavy emphasis on the theoretical basis for much of the science. The more introductory texts that I found were often quite dated and did not offer the blend of theoretical and practical information that I felt was most appropriate for a broad audience of students. I thus developed my own set of lecture notes for these courses, drawing material from a wide range of textbooks and primary literature.
In 1993, I left Chicago to focus my research on the utilization of basic enzymology and protein science for the development of therapeutic agents to combat human diseases. To pursue this goal I joined the scientific staff of the DuPont Merck Pharmaceutical Company. During my first year with this company, a group of associate scientists expressed to me their frustration at being unable to find a textbook on enzymology that met their needs for guidance in laboratory protocols and data analysis at an appropriate level and
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xvi PREFACE TO THE FIRST EDITION
at the same time provide them with some relevant background on the scientific basis of their experiments. These dedicated individuals asked if I would prepare and present a course on enzymology at this introductory level.
Using my lecture notes from Chicago as a foundation, I prepared an extensive set of notes and intended to present a year-long course to a small group of associate scientists in an informal, over-brown-bag-lunch fashion. After the lectures had been announced, however, I was shocked and delighted to find that more than 200 people were registered for this course! The makeup of the student body ranged from individuals with associate degrees in medical technology to chemists and molecular biologists who had doctorates. This convinced me that there was indeed a growing interest and need for a new introductory enzymology text that would attempt to balance the theoretical and practical aspects of enzymology in such a way as to fill the needs of graduate and medical students, as well as research scientists and technicians who are actively involved in enzyme studies.
The text that follows is based on the lecture notes for the enzymology course just described. It attempts to fill the practical needs I have articulated, while also giving a reasonable introduction to the theoretical basis for the laboratory methods and data analyses that are covered. I hope that this text will be of use to a broad range of scientists interested in enzymes. The material covered should be of direct use to those actively involved in enzyme research in academic, industrial, and government laboratories. It also should be useful as a primary text for senior undergraduate or first-year graduate course, in introductory enzymology. However, in teaching a subject as broad and dynamic as enzymology, I have never found a single text that would cover all of my students’ needs; I doubt that the present text will be an exception. Thus, while I believe this text can serve as a useful foundation, I encourage faculty and students to supplement the material with additional readings from the literature cited at the end of each chapter, and the primary literature that is continuously expanding our view of enzymes and catalysis.
In attempting to provide a balanced introduction to enzymes in a single, readable volume I have had to present some of the material in a rather cursory fashion; it is simply not possible, in a text of this format, to be comprehensive in such an expansive field as enzymology. I hope that the literature citations will at least pave the way for readers who wish to delve more deeply into particular areas. Overall, the intent of this book is to get people started in the laboratory and in their thinking about enzymes. It provides sufficient experimental and data handling methodologies to permit one to begin to design and perform experiments with enzymes, while at the same time providing a theoretical framework in which to understand the basis of the experimental work. Beyond this, if the book functions as a stepping-stone for the reader to move on to more comprehensive and in-depth treatments of enzymology, it will have served its purpose.
R A. C
Wilmington, Delaware
‘‘All the mathematics in the world is no substitute for a reasonable amount of common sense.’’
W. W. Cleland
INDEX
Absorption spectroscopy, 189, 205 errors in, 210
Acid-base catalysis, 155, 164 pH effects on, 166
Acid-base chemistry, 29
of amino acid side chains, 45, 48 Activation energy, 27, 152
Active site complementarity, 147 Active site preorganization, 155, 176 Active site structure, 147
Active site titration, 197, 313
Active site triad of serine proteases, 63, 178
Activity staining, in gel electrophoresis, 234
Acyl-enzyme intermediates, 158, 162, 179 Affinity labeling, 346
AIDS, 9, 67
Allosteric constant, 377 Allosteric effectors, 368 Allostery, 367
Alpha carbon, of amino acids, 42 Alpha helix, 58 Alpha-aminoboronate peptides, as
inhibitors of serine proteases, 335 Alpha-amylase, 3
Amino acid sequence, 7, 55 Amino acids, 42
physicochemical properties of, 43 side chain structures of, 44
Amino terminus, 55
Ancient references to enzymes, 2
Anion and polyanion binding in proteins, 50
Antibodies, 178, 233 Apoenzyme, 69
Approximation of reactants, 155 Aromaticity, 20
Arrhenius equation, 28, 249 Arrhenius plots, 250
Aryl azides, 346
Aspartate carbamoyltransferase, 373 Aspirin, as an inhibitor of prostaglandin
synthase, 335 Atomic orbitals, 11 ATPases, 52 Aufbau principle, 14
Autoradiography, 219, 227
Beer’s law, 206 Benzophenones, 346 Beta pleated sheet, 60 Beta turns, 61
Bi bi reactions, 352 Bohr model of atoms, 12
Bond lengths, of peptide components, 53 Bonding and antibonding orbitals, 15 Briggs and Haldane steady state
approach, 115 Bromoacetamido-affinity labels, as
inhibitors of prostaglandin synthase, 336
Bro¨ nsted-Lowry acids and bases, 29, 48 Bro¨ nsted equations, 167
391
392 INDEX
Bro¨ nsted plots, 160, 169 Buffering capacity, 31
Buffers used in enzyme assays, 242 Burst phase kinetics, 159, 196
Carbonic anhydrase, 49 Carboxyl terminus, 56 Carboxypeptidase, 179 Carrier proteins, 260 Catalytic antibodies, 178
Cation and metal binding in proteins, 49 Chemical bonds, 11
Chemical mechanisms of catalysis, 146 Chemical modification, 341
Cheng and Prusoff equations, 285 Chromatography, 102, 224 Chymotrypsin, 63, 179
Cis-prolyl bonds in enzymes, 55 Cis-trans peptide bonds, 54 Coenzymes, see Cofactors Cofactors, 68
effects on velocity, 240 Comformational distortion, 170 Competitive binding, 95 Competitive inhibitors, 273, 358 Compulsory ordered reactions, 354
Computer software for enzyme studies, 387
Concerted transition model of cooperativity, 373
Conjugate bases, 29
Consumer products, use of enzymes in, 1 Continuous assays, 199
Controls, importance of in experimental measurements, 202
Coomassie brilliant blue, 231 Cooperativity, 86, 139, 367
effects on velocity curves, 139, 379 in inhibitor binding, 381
models of, 373 Cooperativity index, 380 Coulombic attractive forces, 32 Coupled reactions, 25, 190 Covalent catalysis, 158 Covalent modification, 50, 341 CPM (Counts per minute), 219 Curie (Ci), 219
Cytochrome c, 189
Cytochrome oxidase, 25, 185, 189
Deadend inhibition, 265, 358 Desalting columns, 224 Detection methods, 204 Digestion, 3
Dihydroorotate dehydrogenase, 9, 185, 190, 220, 235
Dihyrofolate reductase, 292, 300 Dipole moment, 34
Direct assays, 188 Discontinuous assays, 199 Disulfide bonds, 50 Dixon plots, 276, 309 Domains, 65 Dose-response curves, 282
Double displacement reactions, 355 Double reciprocal plots, 90, 128
use in determining inhibitor type, 273
Drugs, enzyme inhibitors as, 8
DPM (disintegrations per minute), 219 DuP697, 339
Eadie-Hofstee plots, 91, 133 Eisenthal-Cornish-Bowden plots, 134 Electron spin, 12
Electronic configuration, of elements common in biological tissue, 15
Electronic state, 22 Electrophilic catalysis, 161 Electrophoresis, 230 Electrostatic interactions, 32 ELISA, 222
End point assays, 199 Enthalpy, 24 Entropy, 24
Enzyme Commission (EC) classification system, 184
Enzyme Data Bank, 186 Enzyme concentration, effects on
velocity, 238
Enzyme reactions, general nomenclature for, 184
Enzyme structure, 5, 42 in inhibitor design, 299
Enzyme, definition of, 4 Enzyme-inhibitor complex, 267 Enzyme-product complex, 113 Enzyme-substrate complexes, 113 Enzymes, as targets for drugs, 8
Equilibrium binding, 76 Equilibrium dialysis, 97
Equilibrium dissociation constant, see
K
Equipment for enzyme studies, 385 E , see Taft steric parameter Excited states, 22
Experimental measures of activity, 188 Extinction coefficient, 206
Feedback loops, in metabolic control, 370
Ficin, 2
Flavins, as cofactors in enzymes, 70 Fluorescence, 104, 211
resonance energy transfer, 213 polarization, 104
quenching, 213 errors in, 216 Flurbiprofen, 336
4,4 -dithioldipyridine, 51 Fractional activity, 283 Free energy ( G), 23
of binding, 77
Free energy diagrams, 27 Freeze-thaw cycling, effects on enzyme
stability, 259
General acid-base catalysis, 164 Glassware, protein adsorption to, 259 Global fitting of inhibition data, 282 Glycoproteins, 52
Glycosylation, 52
Graphic determination of K , 273 for competitive inhibitors, 273 for noncompetitive inhibitors, 278 for uncompetitive inhibitors, 280
GRID program, use in inhibitor design, 302
Ground state, 22
Guanidine hydrochloride, 63
Haldane relationship, 122 Hammett sigma constant, 294 Hanes-Woolf plots, 93, 134 Hemes, as cofactors in enzymes, 70 Hemoglobin, 56, 67, 368
R and T states of, 67, 370 Henderson equation, 311
INDEX 393
Henderson-Hasselbalch equation, 31, 244
Henri-Michaelis-Menten equation, 5, 113
Heterotropic cooperativity, 368 Highest Occupied Molecular Orbital
(HOMO), 23
Hill coefficient, 139, 379 Hill equation, 139, 379 Hill plots, 140
HIV protease, 67 Holoenzyme, 69 Homer’s Iliad, 2 homology modeling, 301
homotropic cooperativity, 367 HPLC (high performance liquid chromatography), 224
Hummel-Dreyer chromatography, 102 Hybrid orbitals, 17
Hydrogen bonding, 33 Hydrophobic interactions, 33 Hydrophobic parameter ( ), 294 Hydrophobicity, 43, 294 Hyperbolic kinetics, 111
deviations from, 137
IC , 96, 282
effects of substrate concentration on, 284
Immunoblotting, 233 Inactivation of enzymes, 260, 320 Index Medicus, 186
Indirect assays, 188 Indomethacin, 337 Induced fit model, 173 Induced strain model, 173
Inhibition, equilibrium treatement of, 268
Inhibitor design, 291 Inhibitor screening, 291 Inhibitors, reversible, 267 Initial velocity, 40, 199
measurements at low substrate concentration, 136
Initiating reactions, 200 Inner filter effect, 216 International units, 257
Ion exchange chromatography, 229 Ion pairs, 32
394 INDEX
Irreversible inactivation, 328, 341 Isobolograms, 289 Isomerization, of enzymes, 320
Isotope effects, in characterization of reaction transition state, 255
Isotope exchange, use in distinguishing reaction mechanism, 360
Isotopes, effects on velocity, 253
k , 114 k /K , 122
K , 76
K , 267
k , 328 Kinases, 51
Kinetic approach to equilibrium, 78 Kinetic perfection, 123
Kinetics, hyperbolic, see Hyperbolic kinetics
Kinetics, sigmoidal, 138, 379 Kinetics, steady state, 115 King-Altman method, 362 K , 118
graphic determination of, 124 k , for slow binding inhibitors, 322
k , 78 k , 78 K , 114
Kyte and Doolittle hydrophobicity index for amino acid residues, 45
Lag phase, 191, 196, 250 Langmuir isotherm, 80 Lewis acids and bases, 29 Ligand, 76
Ligand Binding, 76 methods for measuring, 96
Ligand depletion, 94
Lineweaver Burk plots, see Double reciprocal plots
Lock and key model, 4, 148 Lone pair electrons, 20
Lowest Unoccupied Molecular Orbital (LUMO), 23
Mechanism-based inhibition, 321 Mefanamic acid, 338
Membrane filtration, 99, 224 Metalloproteases, 184, 215
Metals, as cofactors in enzymes, 49, 162 Methotrexate, 292, 300
Methyl thiazolyl tetrazolium, 235 Michaelis Menten equation, see Henri-
Michaelis-Menten equation Microtiter plates, for spectroscopic
assays, 209
Mixed inhibitors, see Noncompetitive inhibitors
Mixing of samples, 200
Molar absorptivity, see Extinction coefficient
Molar refractivity, as a measure of steric bulk, 294
Molecular biology, 7, 56, 172 Molecular dynamics, 301 Molecular orbitals, 15
Monod, Wyman, Changeux model of cooperativity, 373
Morrison equation, 310 Multiple binding sites, 83
equivalent, 83 nonequivalent, 84
Multi-subunit enzymes, 66 Multisubstrate-utilizing enzyme, 350 Mutually exclusive inhibitor binding,
287 Myoglobin, 6, 369
NAD and NADP, as cofactors in enzymes, 25, 71, 190
Native gel electrophoresis, 234 Negative cooperativity, 86, 139, 367 Nicotinamide adenine dinucleotide, see
NAD
Nitroblue tetrazolium, 235 Nitrocellulose, protein binding to, 99,
224, 233
NMR spectroscopy, 7, 266, 299 Nonbonding electrons, 20 Nonspecific binding, 86
Nonsteroidal anti-inflammatory drugs (NSAIDs), 336
Noncompetitive inhibitors, 270 Noncovalent interactions, 32 Nonexclusive binding coefficient, 377 Nucleophilic catalysis, 160
Optical cells, 207
Optical spectroscopy, 104, 205 Orbital angular momentum, 12 Orbital steering, 156
Papain, 2
Partial inhibitors, 272 Dixon plot for, 287
dose-response curves for, 287 Partition coefficient, 294
Pauli exclusion principle, 12 Peak area and peak height, 228 PEG-8000, 260
Peptide bonds, 53 pH, 30
definition of, 30 effects on velocity, 241
induced protein denaturation, 241 Pharmacophore, 291
Phosphatases, 51 Phosphoryl-enzyme intermediates, 51,
162
Phosphorylation, of amino acid residues, 51
Photocrosslinking, 346 Pi bonds, 19
Pi hydrophobicity parameter, 294 Ping-Pong reactions, see Double displacement reactions
pK , 31
graphical determination of, 31 temperature effects on, 243
values of perturbed amino acids, 49, 167, 246
Polarography, 189 Poly-glycine helix, 62 Poly-proline helix, 62 Polypeptide, definition of, 53
Postive cooperativity, 86, 139, 367 Primary structure, 55
Principal quantum number, 12 Product inhibition, 198, 358
use in distinguishing reaction mechanism, 358
Progress curves, 38, 194 kinetic analysis of, 194
Propinquity effect, 156 Prostaglandin synthase, 185, 335
crystal structure of, 336 inhibitors of, 335
INDEX 395
isozymes of, 339
Protein filter binding, 99, 224 Protein folding, 62
Protein precipitation, 223
Proteolytic cleavage sites, nomenclature for, 179
Proton inventory, 256 Proximity effect, 156 Pseudo-first order reactions, 39
Pyridoxal phosphate, as a cofactor in enzymes, 70, 162
QSAR, 295
Quantum numbers, 12 Quantum yield , 212 Quaternary structure, 65
Quinine sulfate, as a quantum yield standard, 213
Quinones, as cofactors in enzymes, 70
R and T states of allosteric proteins, 376 Rack mechanism, 170
Radioactive decay, 36, 219 Radioactivity measurements, 218
errors in, 222 Ramachandran plots, 57 Random coil structure, 62 Random ordered reactions, 352
Rapid equilibrium model of enzyme kinetics, 113
Rapid kinetics, 141
Rapid reaction quenching, 142 Rate constant, 37
Rate enhancement by enzymes, 151 Rates of chemical reactions, 35 Reaction order, 37
Reaction types catalyzed by enzymes, 184
Reagents for enzyme studies, 385 Receptors, 66, 76
Recombinant DNA, see Molecular biology
Reduced mass, 254 Renaturation of proteins after
electrophoresis, 234 Renin, 2, 298
Rennet, 2 Resonance, 20, 54
Resonance energy stabilization, 21
396 INDEX
Retention time, 227 Reversed phase HPLC, 226
Reversible chemical reactions, 39
Salt bridges, 32, 46 Sandwich gel assays, 237 Scatchard plot, 91
Schro¨ dinger wave equation, 12 Scintillation, 219
SDS-PAGE, 230 Secondary plots, 276 Secondary structure, 57 Self-absorption, 216, 222
Selwyn’s test for enzyme inactivation, 261
Semilog plots, 88, 282, 381 of ligand binding, 88
of inhibitor binding, 282 Separation methods, 223 Sequential interaction model of
cooperativity, 374 Serial dilution, 124, 284 Serine proteases, 63, 178, 335
as examples of slow binding inhibition, 335
Sickle cell anemia, 56 Sigma bonds, 17
Sigmoidal kinetics, causes of for noncooperative enzymes, 382
Site directed mutagenesis, see Molecular biology
Size exclusion chromatography, 102, 224, 229
Sliver staining, 231
Slow binding inhibitors, 318 determining mechanism of, 325 determining mode of interaction with
enzyme for, 330
determining reversibility of, 332 preincubation of with enzymes, 324 progress curves for, 321
Slow, tight binding inhibitors, 323 Solvent isotope effects, 255 Spacial probability distribution of
electrons, 12
Specific acid-base catalysis, 164 Specific activity, 257
Specific binding, 86 Specific radioactivity, 220
Spectroscopic methods, 5, 104, 205 in ligand binding, 104
in enzyme assays, 205 Stability of enzymes, 258 Statine, 298
Steady state kinetics, 115 Steric bulk, 52
Stokes shift, 212 Stopped-flow, 142 Stopping reactions, 200
Storage conditions for enzymes, 258 Stromelysin, 184, 185, 216 Structural complementarity, 147, 299
between competitive inhibitors and active site, 299
between substrate and active site, 147 Structure-activity relationship (SAR),
291
Structure-based inhibitor design, 147 Substrate concentration, effect of on
velocity, 111, 198
Substrate depletion, effects on velocity, 110
Substrate inhibition, 137 Substrate protection, 344 Substrate specificity, 122, 147, 171 Subtilisin, 179
Subunits, 65 Super-secondary structure, 64
Surface plasmon resonance, 267
Taft steric parameter, 293 Temperature, effects on velocity, 28, 248 Tertiary structure, 62
Thermal denaturation of proteins, 248 Thermodynamics, of chemical reactions,
23
Three point attachment model, 149 3 helix, 61
Threonine deaminase, 372 Tight binding inhibitors, 305
determining K of, 310 distinguishing inhibitor type for, 307 IC values for, 306
use in determining active enzyme concentration, 313
Time course, of enzyme reactions, see Progress curves
Time dependent inhibitors, 318