handbook of biochemical kinetics a guide to dynamic processes in the molecular life sciences,...

Handbook of Biochemical Kinetics

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Handbook of Biochemical KineticsDaniel L. Purich R. Donald Allison

Department of Biochemistry and Molecular Biology University of Florida College of Medicine Gainesville, Florida

ACADEMIC PRESSSan Diego London Boston New York Sydney Tokyo Toronto

This book is printed on acid-free paper. Copyright 2000 Academic Press All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher. Requests for permission to make copies of any part of the work should be mailed to the following address: Permissions Department, Harcourt Brace & Company, 6277 Sea Harbor Drive, Orlando, Florida 32887-6777. Academic Press A Division of Harcourt Brace & Company 525 B Street, Suite 1900, San Diego, CA 92101-4495 http://www.apnet.com Academic Press 2428 Oval Road, London NW1 7DX http://www.hbuk.co.uk/ap/ Library of Congress Catalog Card Number: 99-63958 International Standard Book Number: 0-12-568048-1 Printed in the United States of America 99 00 01 02 03 04 MM 9 8 7 6 5 4 3 2 1

Table of ContentsPreface.............................. vii Abbreviations & Symbols.......................... xi Abbreviated Binding Schemes .............. xxi Source Words......................1 Wordnder .....................717


v


PrefaceThe biotic world is doubtlessly the best known example of what Nobelist Murray Gell-Mann has termed complex adaptive systemsa name given to those systems possessing the innate capacity to learn and evolve by utilizing acquired information. Those familiar with living systems cannot but marvel at each cells ability to grow, to sense, to communicate, to cooperate, to move, to proliferate, to die and, even then, to yield opportunity to succeeding cells. If we dare speak of vitalism, especially as the new millennium is eager to dawn, we only do so to recognize that homeostatic mechanisms endow cells with such remarkable resilience that early investigators mistook homeostasis as a persuasive indication that life is self-determining and beyond the laws of chemistry and physics. A shared goal of modern molecular life scientists is to understand the mechanisms and interactions responsible for homeostasis. One approach for analyzing the mechanics of complex systems is to determine the chronology of discrete steps within the overall processa pursuit called kinetics. This strategy allows an investigator to assess the structural and energetic determinants of transitions from one step to the next. By identifying voids in the time-line, one considers the possibility of other likely intermediates and ultimately identies all elementary reactions of a mechanism. Kinetics is an analytical approach deeply rooted in chemistry and physics, and biochemists have intuitively and inventively honed the tools of chemists and physicists for experiments on biological processes. Biochemical kinetics rst began to ourish in enzymologya eld which has gainfully exploited advances in physical organic chemistry, structural chemistry, and spectroscopy in order to dissect the individual steps comprising enzyme mechanisms. No apology is offered, nor should any be required, for our strong emphasis on chemical kinetics and enzyme kinetics. Scientists working within these disHandbook of Biochemical Kinetics ciplines have enjoyed unparalleled success in dissecting complex multistage processes. Mechanisms are tools for assessing current knowledge and for designing better experiments. As working models, mechanisms offer the virtues of simplicity, precision, and generativity. Simplicity arises from the symbolic representation of the interactions among the minimal number of components needed to account for all observed properties of a system. Precision emerges by considering how rival models have nonisomorphic features (i.e., testable differences) that distinguish one from another. Generativity results from the recombining of a models constituent elements to admit new ndings, to predict new properties, and to stimulate additional rounds of experiment. For chemical and enzyme kineticists, the goal of this recursive enterprise is to determine a mechanism (a) that accounts for responses to changes in each components concentration, (b) that explains the detailed time-evolution of all chemical events, (c) that denes the concentration and structure of transient intermediates, (d) that makes sense of relevant changes in positional properties (i.e., conformation, conguration, and/or physical location), and (e) that reconciles the thermodynamics of all reactions steps and transitions. In this respect, the rigor of chemical and enzyme kinetics teaches us all how best to invent new approaches that appropriately balance theory and experiment. The inspiration for this HANDBOOK stemmed from our shared interest in teaching students about the logical and systematic investigation of enzyme catalysis and metabolic control. We began twenty-ve years ago with the teaching of graduate-level courses (Chemical Aspects of Biological Systems; Enzyme Kinetics and Mechanism) at the University of California Santa Barbara as well as a course entitled Enzyme Kinetics at the Cornell University Medical College. More recently, we have vii

Prefacetaught undergraduate students (A Survey of Biochemistry and Molecular Biology) as well as graduate students (Advanced Metabolism; Physical Biochemistry and Structural Biology; Dynamic Processes in the Molecular Life Sciences) here at the University of Florida. Our lectures have included material on the theory and practice of steady-state kinetics, rapid reaction kinetics, isotope-exchange kinetics, inhibitor design, equilibrium and kinetic isotope effects, protein oligomerization and polymerization kinetics, pulse-chase kinetics, transport kinetics, biomineralization kinetics, as well as ligand binding, cooperativity, and allostery. Because no existing text covered the bulk of these topics, we resorted to developing an extensive set of lecture notesan activity that encouraged us to consider writing what we initially had envisioned as a short textbook on biochemical kinetics. What also became clear was that, before and during any detailed consideration of a molecular process, teachers must always take pains to explain the associated terminology adequately. In 1789, the French chemist Antoine Lavoisier aptly declared: Every branch of physical science must consist of the series of facts that are the objects of the science, the ideas that represent these facts, and the words by which these ideas are expressed. And, as ideas are preserved and communicated by means of words, it necessarily follows that we cannot improve the science without improving language or nomenclature. We recognized that there was no published resource to help students come to grips with the far-ranging terminology of biochemical kinetics. Furthermore, as the distinction between scientic disciplines becomes blurred by what may be called the interdisciplinary imperative, students and practicing scientists from other disciplines will require a reliable sourcebook that explains terminology. Far too much time is wasted when students trace a nger over many pages of a textbook, only to nd a partial denition for a sought-after term. Moreover, as more bioscientists come from countries not using English as a working language, there is an even greater need for a reliable and clearly written sourcebook of denitions. A dictionary format became an appealing possibility for our HANDBOOK, but we also wished to treat many terms in greater depth than found in any dictionary. This led us to adopt the present word-list format which in many viii respects resembles the Micropaedia section of the ENCYCLOPAEDIA BRITANNICA. One loses the seamless organization that can be realized in multichapter expositions that systematically develop a series of topics. We have accordingly attempted to mitigate this problem by including longer tracts on absorption and uorescence spectroscopy, biomineralization, chemical kinetics, enzyme kinetics, Hill and Scatchard treatments, ligand binding cooperativity, kinetic isotope effects, and protein polymerization. Likewise, we have extensively inserted cross-references at appropriate locations within many entries. One intrinsic advantage of a mini-encyclopedia, however, is that in subsequent printings we should be able to make corrections and additions, or even deletions of an entire term, without upsetting the overall format. We also felt that readers should be encouraged to consult the most authoritative sources on particular topics. For this reason, we developed a collection of over 5000 literature references and, in many cases, our citations credit the original papers on a given topic. We have included the names of nearly 1000 enzymes, along with chemical reactions, EC numbers, and, in many instances, their biochemical and catalytic properties. The references cited are not intended to be comprehensive; rather, they serve to guide the reader to further interesting and helpful reading on subjects we have discussed. Where possible, at least one reference is included to provide information on assay protocols for the listed enzyme. We also urge readers to use the Wordnder (included at the back of the Handbook) to take fullest advantage of the text and reference material. The nearly 8000 entries in the Wordnder represent all listed source words as well as other subheadings, keywords, or synonyms. Each entry is immediately followed by the recommended source entries, and many source words are also cross-referenced to guide the reader to other related source words. One of us (D.L.P.) has been a member of the METHODS ENZYMOLOGY family of editors for well over two decades. The volumes in this series on Enzyme Kinetics and Mechanism have become a standard for those interested in biological catalysis. As the form of this book began to emerge, we quickly recognized that METHODS IN ENZYMOLOGY could serve as an additional source for annotations on recommended theories and practices for kinetic studies on a wide range of topics. The Handbook contains nearly 6000 METHODS IN ENZYMOLOGY citaIN


Prefacetions, and we have indicated the topic, volume, and beginning page for each at the foot of many of the source words. We trust that users of our Handbook will benet from improved access to the rst 280 volumes of METHODS IN ENZYMOLOGY. For the derivations presented in this Handbook, we have assumed that the reader is familiar with the fundamentals of differential and integral calculus. To those who are loathe to engage in the rigor of mathematics, we say Take heart! The successful study of kinetics requires only that students work out a considerable number of problems which are both theoretical and numerical in character. We are reminded that Mithridates VI, the Grecian king of Pontus, is said to have acquired a tolerance to poison by taking gradually increasing doses. To aid those seeking their own intellectual mithridate (i.e., acquired antidote), we provide scores of step-by-step derivations and practical advice on how to derive particular rate expressions. Likewise, we have included detailed protocols for H. J. Fromms systematic theory-of-graphs method as well as W. W. Clelands net reaction rate method. We are also greatly indebted to Dr. Charles Y. Huang for permitting us to include entire tracts from his chapter (which originally appeared in Volume 63 of METHODS IN ENZYMOLOGY) on the derivation of initial velocity and isotope exchange rate equations. We immediately recognized how daunting the task would be to attempt to surpass Dr. Huangs truly outstanding treatment. The success of our HANDBOOK OF BIOCHEMICAL KINETICS can only be judged by those using this manual for some period of time. We have come to recognize that we could not possibly represent all of the topics falling within the realm of biochemical kineticsand certainly not within a rst edition. We are also certain that, despite a determined effort to cover the terminology of chemical and enzyme kinetics, we have still overlooked some important issues. We had also hoped to include additional kinetic techniques applied in pharmacokinetics, cell biology, electrophysiology, and metabolic control analysis. Time constraints also prevented our developing mathematical sections on Laplace transforms, vector algebra, distribution functions, and especially statistics. Eventually, we aspire to create a compact disk version of this Handbook, appropriately presented as hyperlinked text; that same CD should have room for selected problems/ exercises along with step-by-step solutions, as well as down-loadable programs for kinetic simulation, algorithms for symbolic derivation of rate equations, molecular dynamics and related modeling techniques, and tried-and-true statistical methods. We also hope that our readers will not hesitate to advise us of shortcomings, missed terms, as well as techniques meriting denition, mention, or further explanation. We shall be forever grateful for such guidance. We thank our students and colleagues for reading earlier drafts of our manuscript, and we are especially grateful to both Shirley Light and Dolores Wright of Academic Press for their insights, help, and thorough editing of the text. We also thank Academic Press for allowing us to incorporate the numerous annotations to METHODS IN ENZYMOLOGY. Finally, as rst- and second-generation disciples of Professor Herbert J. Fromm, we dedicate this book to him, in recognition of his germinal and indelible contributions to the eld of enzyme kinetics and mechanism. Daniel L. Purich R. Donald Allison January, 1999


ix


Abbreviations & SymbolsRoman Letters and SymbolsA A* A Molecule in the ground state Acceptor molecule (in uorescence) Molecule in the excited state SI symbol for absorbance (unitless) SI symbol for Helmholtz energy ( J ) SI symbol for the pre-exponential term in Arrhenius equation (mol 1m3)n 1 s 1 Change in absorbancy Percent absorption of light (100 %T) Concentration of A D Ci or ci c or Ci C or c C Cp Molar concentration (M or moles/L) SI symbol for heat capacity ( J K Constant pressure standard heat capacity per mole Weight concentration of the ith species Symbol for Curie (old unit of radioactivity) SI unit for speed of light in a vacuum; (value 2.998 108 m s 1) SI unit for speed of light in a medium (m s 1) Concentration SI symbol for debye (unitless) Donor molecule (in uorescence) SI symbol for translational diffusion constant (m2 s 1) Spectroscopic energy of dissociation of a diatomic molecule in the Morse equation Difference in dissociation energies of products and reactants measured from zero-point energies Dalton Rotational diffusion constant Density extrapolated to 20 C, water Density Collision diameter Free or uncomplexed enzyme General symbol for enzyme Effector molecule xi1

)

A %A

co

c

[A]

A, B, C, . . . Substrate A, B, C, . . . A, B, C, . . . Coulombic contributions to the potential energy of interaction Moments of inertia of transition-state complex Aij A ai ao B B Amplitude of kinetic decay Angstrom unit (1010

D

m) Doo

Thermodynamic activity of species i Bohr radius Generalized base e2 /2 kT or [e3/( kT)2/3](2 No / 1000)1/2, a constant in the DebyeHuckel limiting law Second virial coefcient for the mutual interactions of species i and j Becquerel (unit of radioactivity disintegration per second) Coulomb 1

Da Drot d20,w d

Bi,j Bq C

E


Abbreviations & SymbolsE Energy Initial kinetic energy of relative motion of reactants Electric vector of light Activation energy Total enzyme concentration Equivalent Equilibrium Standard electromotive force Energy of molecule in excited singlet state Torsional potential energy Exponential function Charge on an electron (value 1.602 10 19 coulombs) Equatorial position of a substituent on a molecule Pseudo-equatorial position of a substituent on a molecule Modied enzyme form in ping pong mechanisms Force Free energy (archaic) Fluorescence Rotational-vibrational energy distribution function Momentum distribution function Faraday Fugacity Number of sites for acceptor on ligand (so-called ligand valence) Oscillator strength Translational frictional coefcient Fractional attainment of isotopic equilibrium Fluorescencesample/Fluorescencestandard Gibbs free energy Gravitational constant i J H

G

Standard Gibbs free energy change per mole Standard Gibbs free energy of activation Gram Degeneracy of the lower state Degeneracy of the upper state Henry (unit of self-inductance and mutual inductance) Enthalpy Hamiltonian Magnetic eld strength at nucleus of a molecule Standard enthalpy change per mole Standard enthalpy of activation Magnetic eld Magnetic eld intensity at which resonance takes place Hertz (unit of frequency cycles per second) Plancks constant 6.626 10 J sec or 6.626 10 27 erg sec h/2 1.055 10 27 erg sec 1034 34

G

E Ea [Eo] or Eo Eq or eq

g ge gu H

E Es Etor e e

H

Hlocal

H

H Hres Hz

e

F

F F

h

J sec or 1.055

I

Nuclear spin quantum number Inhibitor Intensity of radiation Ionic strength Light transmittance Inhibitor yielding 50% inhibition or 0.5 the uninhibited rate Intensity of light at wavelength Intensity of emitted light at wavelength Square root of ( 1) Joule Handbook of Biochemical Kinetics

F f

I

I50 or I0.5 I( ) I( )f

f

Frel G

xii

Abbreviations & SymbolsJ Nuclear magnetic resonance coupling coefcient Flux density (units particles area 1 time 1) Apparent order of a binding reaction Symbol for Kelvin Macroscopic equilibrium constant Acid dissociation constant KE L L KD KF Dissociation constant Formation constant (synonym of association constant) Dissociation constant for ligand F for an allosteric protein Macroscopic inhibition constant Macroscopic ionization constant L L M M M MnMr Mwm me N No n n n * *

kd

Intrinsic dissociation constant (reciprocal of ki,j)

kiH , kiD , kiT A rate constant for isotopic isomers containing H, D, or T kH/kD ki,j Kinetic isotope effect Intrinsic association or binding constant (reciprocal of kd) for interaction between sites on species i and j Kinetic energy Liter Avogadros number Angular momentum Allosteric constant equal to [To]/[Ro] Molecular weight Molar Transition-state complex Magnetization Number average molecular weightRelative molecular mass Weight average molecular weightMeter Mass of electron at rest value 9.1094 10 28 g Newton (unit of force) Avogadros number 1023 mol 1 Refractive index Number of moles Electronic transition Electronic transition Hill coefcient Generalized symbol for product Pressure log10 Ka Oxygen partial pressure 6.0221

j K K or Keq Ka

KA , KB , . . . Dissociation constant for ligand A, B, C, . . . Kap Apparent equilibrium constant

Ki

Kia , Kib , . . . Dissociation constants in enzyme kinetics KR KS KT Dissociation constant for ligand that binds to R-state of allosteric protein Equilibrium constant for dissociation of ES complex Dissociation constant for ligand that binds to T-state of an allosteric protein The constant equal to the product of [H ] (or, [H3O ]) and [OH ] in an aqueous solution

Kw

K1 , K2 , K3 , . . . Stepwise binding or dissociation constants for successive attachments of ligand to an oligomeric receptor k or kB k Boltzmann constant Rate constant Microscopic equilibrium constant Catalytic constant; turnover number Specicity constant

nH or nHill P P or p pKa pO2

kcat kcat/Km


xiii

Abbreviations & Symbols(pO2 )0.5 Q Q QCO2 Qsyn q qo R Oxygen partial pressure at 0.5 saturation Coulomb (unit of electrostatic charge) Heat absorbed by a dened systemt1/2 T1 T2 t Longitudinal relaxation time Transverse relaxation time Time Half-life Internal energy Volume Volume of activation Hydrated volume Maximal velocity

Amount CO2 released by tissue Synergism quotient Quantum yield Unquenched quantum yield Universal gas constant Electric resistance Gross rate of isotopic exchange Rydberg constant Fraction of allosteric protein in the R-state Radius of gyration Radius Distance of separation Polymer end-to-end vector Svedberg unit (1013

U V V Vh Vm or Vmax

R R RG r

Vm,f or Vmax,f Maximal velocity in the forward direction Vm,r or Vmax,r Maximal velocity in the reverse direction V/K v Ratio of Vmax to Km Speed Initial velocity of enzyme-catalyzed reaction Vibrational frequency dX/dt Equilibrium concentration of substance XiDifference between temporal and equilibrium concentration of Xi Charge on a macromolecule or ion in units of e

r S SA SA S

s) X (Xi)(Xi) z

Partial molal entropy Unitary part of the partial molal entropy Standard entropy change Standard entropy of activation Scattering vector Singlet state Second (unit of time) Sedimentation coefcient Equilibrium constant for helix growth Sedimentation coefcient corrected to 20 C, water Unit vector along scattered radiation Temperature Percent transmission of light Melting temperature

S

S S1 s s

Greek Letters and SymbolsDegree of association Alpha particle Electric polarizability Reduced concentration ([F]/KF) for allosteric proteinH

s20,w s T %T Tmxiv

Hill coefcient Reduced concentration ([I]/KI) for allosteric protein

e

Bohr magneton


Abbreviations & SymbolsSurface concentration (mol m 2) Parameter affecting relaxation amplitude Reduced concentration ([A]/KA) for allosteric protein Phase shift Chemical shift in nuclear magnetic resonance Torque Molar absorptivity Dielectric constantn,i

(r)

Electron density Summation algorithm Relaxation time (t) Lag time (s) Phi relation in enzyme kinetics Electrical potential Quantum yield (unitless) Mole fraction of component I Solid angle Statistical factor for ligand-i binding at n sites on a macromolecule Angular momentum Circular frequency (Hz) Ionic strength (archaic)

Ellipticity in circular dichroism Solution viscosityo

Solvent viscosity Intrinsic viscosityo

[ ]i

Larmor frequency (Hz) Angular velocity (rad s1

Fraction of ligand saturation of ith site Molar ellipticity Transmission coefcient for transition state Inverse screening length Wavelength Kinetic decay time

or s 1)

[ ]

Mathematical Symbols, , f f Directional angles First derivative Second derivative Partial derivative, Jacobian Integral Average Overlap interval Expectation value integral * Superscript designating radioactive substance Superscript designating excited state Subscript designating complex conjugate Superscript for transition state Partial differential of x with respect to time at constant y Vector differential or gradient, ix jy k z

i

Chemical potential of ith species per mole Standard chemical potential per mole Magnetic moment Ionic strength Electric dipole moment operator Reduced mass, mAmB/(mA mB) Frequency Fractional saturation of ligand binding sites Grand partition function in the Wyman treatment Product algorithm Osmotic pressure Density (mass per unit volume)

i

o

m

( x/ t)y


xv

Abbreviations & Symbols2 22

Second derivative operator,2

ACP ADA Ade ADH ADP Ala ALA AMP amu Arg Asn Asp Asx ATCase ATP B BES Bi bp BPG BPTI Bq C CaM cAMP CAP cAPK CAPS

Acyl carrier protein Adenosine deaminase Adenine Alcohol dehydrogenase Adenosine 5 -diphosphate Alanine or alanyl -Aminolevulinic acid or aminolevulinate Adenosine 5 -monophosphate Atomic mass unit (1.66 1.66 10 24 g) Arginyl or arginyl Asparagine or asparaginyl Aspartic acid, aspartate, or aspartyl Aspartate asparagine or aspartyl asparaginyl Aspartate transcarbamoylase Adenosine 5 -triphosphate Aspartate asparagine (or aspartyl asparaginyl) N, N-Bis(2-hydroxyethyl)-2aminoethanesulfonic acid Two-substrate enzyme system Base pairD-2,3-Bisphosphoglycerate

x

2

y

2

z2 Constant time interval () (,) [] [,] Activity of a solute Open interval Concentration of a solute Closed interval Innite dilution, typically as a subscript

10

27

kg or

Multiples/Submultiples1012 9

Tera (symbol Giga (symbol Mega (symbol Kilo (symbol Deci (symbol Centi (symbol Milli (symbol Micro (symbol Nano (symbol Pico (symbol Femto (symbol Atto (symbol Zepto (symbol

T) G) M) k) d) c) m) m) n) p) f) a) z)

10

106 103 10 10 10 10 10 10 10 10 101 2 3 6 9 12 15 18 21

Bovine pancreatic trypsin inhibitor Becquerel Cytosine Cysteine or cysteinyl Calmodulin Cyclic AMP Catabolite gene activating protein Protein kinase A (or cyclic AMPstimulated protein kinase) 3-(Cyclohexylamino)propanesulfonic acid Handbook of Biochemical Kinetics

Biochemical AbbreviationsA aa aaRS ACAT ACES AChxvi

Adenine Alanine or alanyl Amino acid Aminoacyl-tRNA Acyl-CoA:cholesterol acyltransferase N-(2-Acetamido)-2aminoethanesulfonic acid Acetylcholine

Abbreviations & SymbolsCbzcDNA CDP CHES Chl CM cmc CMP CoA CoASH CoQ CTP Cys D d dd DEAE DFP DG DHAP DHF DHFR DMF DMS DMSO DNP DolL-DOPA

BenzyloxycarbonylComplimentary strand DNA Cytidine 5 -diphosphate 3-(Cyclohexylamino)ethanesulfonic acid Chlorophyll Carboxymethyl Critical micelle concentration Cytidine 5 -monophosphate Coenzyme A Coenzyme A Coenzyme Q Cytidine 5 -phosphate Cysteine or cysteinyl Dalton Aspartic acid, aspartate, or aspartyl Deoxy Dideoxy Diethylaminoethyl Diisopropyl uorophosphate sn-1,2-Diacylglycerol Dihydroxyacetone phosphate Dihydrofolate Dihydrofolate reductase Dimethylformamide Dimethyl sulfate Dimethylsulfoxide 2,4-Dinitrophenyl DolicholL-3,4-Dihydroxyphenylalanine

EDTA EF EGTA

Ethylenediaminetetraacetic acid or its conjugate base Elongation factor Ethylene glycol bis( -aminoethyl ether)-N,N,N ,N -tetraacetic acid or its conjugate base N-2-Hydroxyethylpiperazinepropanesulfonic acid (also known as HEPPS)

EPPS

EPR or ESR Electron paramagnetic resonance or Electron spin resonance F FAD FADH FADH2 FBP Fd fMet FMN F1P F6P G GABA Gal GalNAc GAP GDP Gla Glc Gln Glu Gly GMP Phenylalanine or phenylalanyl Oxidized avin adenine dinucleotide Radical form of reduced avin adenine dinucleotide Reduced avin adenine dinucleotide Fructose 1,6-bisphosphate Ferredoxin N-Formylmethionine Flavin mononucleotide Fructose 1-phosphate Fructose 6-phosphate Guanine Glycine or glycyl -Aminobutyric acid Galactose N-Acetylglucosamine Glyceraldehyde 3-phosphate Guanosine 5 -diphosphate 4-Carboxyglutamic acid or 4-carboxyglutamyl Glucose Glutamine or glutaminyl Glutamic acid, glutamate, or glutamyl Glycine or glycyl Guanosine 5 -monophosphate xvii

DPN DSC E

see recommended abbreviation NAD Differential scanning calorimetry Glutamic acid, glutamate, or glutamyl


Abbreviations & SymbolsG1P G6P GSH GSSG GTP H HA Hb HbA HbCO HbO2 HbS HbF HDL HEPES Glucose 1-phosphate Glucose 6-phosphate Glutathione (sometimes referred to as reduced glutathione) Glutathione disulde (sometimes referred to as oxidized glutathione) Guanosine 5 -triphosphate KF Histidine or histidyl Hemagglutinin Hemoglobin Adult hemoglobin Carbon monoxide hemoglobin Oxyhemoglobin Sickle cell hemoglobin Fetal hemoglobin High density lipoprotein N-2-Hydroxyethylpiperazine-N -2ethanesulfonic acid (also written as Hepes) See EPPS Hypoxanthine:guanine phosphoribosyltransferase Histidine or histidyl -Hydroxymethylglutaryl-CoA Heterogenous nuclear RNA Heat shock protein Hydroxyproline Isoleucine or isoleucyl Intermediate density lipoprotein Initiation factor Immunoglobulin Isoleucine or isoleucyl Inosine 5 -monophosphate Isopropylthiogalactoside NAG NAM NANA L LCAT LDH LDL Leu Lys M Klenow factor Leucine or leucyl Lecithin:cholesterol acyl transferase Lactate dehydrogenase Low density lipoprotein Leucine or leucyl Lysine or lysyl Methionine or methionyl IR IS ITP K kb kD or kDa Infrared Insertion sequence Inosine 5 -triphosphate Lysine or lysyl Kilobase pair Kilodalton

MALDI-MS Matrix-assisted laser desorption ionization-mass spectroscopy Man Mb MbCO MbO2 MES Met MetHb MetMb MOPS MS N NAD NADH Mannose Myoglobin Carbon monoxide myoglobin Oxymyoglobin 2-(N-Morpholino)ethanesulfonic acid Methionine or methionyl Methemoglobin (or, Fe(III)Hb) Metmyoglobin (or, Fe(III)Mb) 3-(N-Morpholino)propanesulfonic acid Mass spectroscopy/spectrometry Asparagine or asparaginyl Oxidized nicotinamide adenine dinucleotide Reduced nicotinamide adenine dinucleotide N-Acetylglucosamine N-Acetylmuramic acid N-Acetylneuraminic acid Handbook of Biochemical Kinetics

HEPPS HGPRT His HMG-CoA hnRNA hsp Hyp I IDL IF IgG Ile IMP IPTG xviii

Abbreviations & SymbolsNMN NMR NOESY Nicotinamide mononucleotide Nuclear magnetic resonance Nuclear Overhauser effect spectroscopy Nucleoside 5 -triphosphate Proline or prolyl Phosphate Phosphoenolpyruvate Phosphofructokinase Prostaglandin 2-Phosphoglycerate 3-Phosphoglycerate Phosphoglucoisomerase Phosphoglucomutase Phenylalanine or phenylalanyl Phosphatidylinositol 4,5-bisphosphate Piperazine-N,N -bis(2-ethanesulfonic acid) Pyruvate kinase Protein kinase A (or cyclic AMPstimulated protein kinase) Protein kinase C Phenylketonuria Pyridoxal 5-phosphate DNA polymerase Inorganic pyrophosphate Proline or prolyl Prion protein 5-Phosphoribosyl- -pyrophosphate Photosystem Glutamine or glutaminyl Ubiqinone (Coenzyme Q or CoQ) THF Thr TAPS TCA PKC PKU PLP Pol PPi Pro PrP PRPP PS Q Ter TES SAM Ser T QCO2 The amount (in microliters) of CO2 given off (under standard conditions of pressure and temperature) per milligram of tissue per hour Quasi-elastic laser light scattering Ubiqinol Four substrate enzyme system Arginine or arginyl Ribo Ribose 5-phosphate Reverse phase chromatography Reverse transcriptase Receptor tyrosine kinase Ribulose 1,5-bisphosphate Ribulose 5-phosphate Serine or seryl Svedberg constant S-Adenosylmethionine Serine or seryl Threonine or threonyl Thymine Tris(hydroxymethyl)methylaminopropanesulfonic acid Tricarboxylic acid Three-substrate enzyme system N-Tris(hydroxymethyl)methyl-2aminoethanesulfonic acid Tetrahydrofolate Threonine or threonyl

QELS QH2 Quad R r R5P RPC RT RTK Ru1,5P2

NTP P P (or p) PEP PFK PG 2PG 3PG PGI PGM Phe PIP2 PIPES

Ru5P S

PK PKA

TIM or TPI Triose-phosphate isomerase TPP Tris TS TX Thiamin pyrophosphate (or thiamin diphosphate) Tris(hydroxymethyl)aminomethane Thymidylate synthase Transition state Transition state intermediate xix


Abbreviations & SymbolsTyr U Uni V Val X Tyrosine or tyrosyl Uridine One-substrate enzyme system Valine or valyl Valine or valyl Nonstandard or unknown amino acid or amino acyl XAFS Y YADH Z Xaa Unspecied amino acid or amino acyl residue X-ray analysis for structure Tyrosine or tyrosyl Yeast alcohol dehydrogenase Glutamate glutaminyl glutamine or glutamyl

xx


Abbreviated Binding SchemesThe following diagrams indicate the binding interactions for enzyme kinetic mechanisms. To conserve space, the notation used in this Handbook is a compact version of the diagrams rst introduced by Cleland1. His diagram for the Ordered Uni Bi Mechanism is as follows:EA-to-EX and EX-to-EPQ reversible:E

A

(EA}EX}EPQ)

P (EQ)

Q

E

EA-to-EX reversible and EX-to-EPQ irreversible:E

A

(EA}EX

EPQ)

P (EQ)

Q

E

EA-to-EX irreversible and EX-to-EPQ irreversible:E

A

(EA

EX

EPQ)

P (EQ) Q

E

Throughout this handbook, we have used the following single-line, compact notation:

For the case of so-called iso mechanisms, the compact diagrams are as follows:E

A (EA}EX

EPQ) P Q

F}E

where F}E represents the reversible isomerization step.E

A

(EA}EPQ)

P (EQ)

Q

E

This convention offers the advantage that it can be readily reproduced on virtually all word processors and typesetting devices without creating any special artwork. The enzyme surface is represented by the underline, in this case preceded by a subscript E to indicate the unbound (or free) enzyme before any substrate addition and after all products desorb. An arrow pointing to the line indicates binding, and the reader should understand that reversible binding is taken for granted. Moreover, unlike the original Cleland diagram, product release always is indicated by a downward arrow, because this systematic usage emphasizes the symmetry of certain mechanisms. The symbol A indicates that substrate A adds; the symbol A or B , A or B or C , etc., indicates random addition of two or three substrates, respectively. Interconversions of enzyme-bound reactants can be reversible (}) or irreversible ( ).

Other examples of one-substrate and two-substrate kinetic mechanisms include: Ordered Uni Bi MechanismE

A

(EA}EPQ)

P (EQ)

Q

E

Random Uni Bi MechanismE

A

(EA}EPQ )

P or Q

E

Random Bi Uni MechanismE

A or B

(EAB}EP)

P

E

Ordered Bi Uni MechanismE

A

(EA)B (EAB}EP)

P

E

Ordered Bi Bi MechanismE

A

(EA) B

(EAB}EPQ)

P (EQ)

Q

E

Taking the Ordered Uni Bi mechanism as an example, we can consider several additional possibilities:Handbook of Biochemical Kinetics

Ordered Bi Bi Theorell-Chance MechanismE

A

(EA) B

P (EQ)

Q

E

xxi

Abbreviated Binding SchemesPing Pong Bi Bi MechanismE1

Random Bi Bi Mechanism

A

(EA}FP)

P (F) B

(FB}EQ)

Q

E

E

A or B

(EAB}EPQ )

P or Q

E

W. W. Cleland (1963) Biochem. Biophys. Acta. 67, 104.

xxii


Source WordsAA, B, C, . . ./P, Q, R, . . .Symbols for substrates and products, respectively, in multisubstrate enzyme-catalyzed reactions. In all ordered reaction mechanisms, A represents the rst substrate to bind, B is the second, etc., whereas P denotes the rst product to be released, Q represents the second, etc. See Cleland Nomenclature known actin regulatory proteins led to the identication of distinct Actin-Based Motility (or Actin-Based-Motor) homology sequences:ABM-1: (D/E)FPPPPX(D/E) [where X ABM-2: XPPPPP [where X P or T]

G, A, L, P, or S]

AB INITIO MOLECULAR-ORBITAL CALCULATIONSA method of molecular-orbital calculations for determining bonding characteristics and other structural information about a wide variety of compounds and molecular congurations, including those that may not be directly observable (e.g., transition state congurations with partial bonds). Although ab initio calculations are typically applied to systems with a small number of atoms, these computationally intensive calculations can be helpful in providing insights about the enzyme-catalyzed reactions. Related methods, known as semiempirical methods, use simplifying assumptions in the calculations and are determined more quickly than standard ab initio methods.W. J. Hehre, L. Radom, P. von R. Schleyer & J. A. Pople (1986) Ab Initio Molecular Orbital Theory, Wiley, New York. T. Clark (1985) A Handbook of Computational Chemistry, Wiley, New York. W. G. Richards & D. L. Cooper (1983) Ab Initio Molecular Orbital Calculations for Chemists, 2nd ed., Oxford Press, Oxford. W. Thiel (1988) Tetrahedron 44, 7393.

Actin-based motility involves a cascade of binding interactions designed to assemble actin regulatory proteins into functional locomotory units. Listeria ActA surface protein contains a series of nearly identical EFPPPP TDE-type oligoproline sequences for binding vasodilator-stimulated phosphoprotein (VASP). The latter, a tetrameric protein with 20-24 GPPPPP docking sites, binds numerous molecules of prolin, a 15 kDa regulatory protein known to promote actin lament assembly2. Laine et al.3 recently demonstrated that proteolysis of the focal contact component vinculin unmasks an ActA homologue for actin-based Shigella motility. The ABM-1 sequence (PDFPPPPPDL) is located at or near the C-terminus of the p90 proteolytic fragment of vinculin. Unmasking of this site serves as a molecular switch that initiates assembly of an actin-based motility complex containing VASP and prolin. Another focal adhesion protein zyxin4 contains several ABM-1 homology sequences that are also functionally active in reorganizing the actin cytoskeletal network.1

D. L. Purich & F. S. Southwick (l997) Biochem. Biophys. Res. Comm. 231, 686. 2 F. S. Southwick & D. L. Purich (1996) New Engl. J. Med. 334, 770. 3 R. O. Laine, W. Zeile, F. Kang, D. L. Purich & F. S. Southwick (1997) J. Cell Biol. 138, 1255. 4 R. M. Golsteyn, M. C. Beckerle, T. Koay & E. Friedrich (1997) J. Cell Sci. 110, 1893.

ABM-1 & ABM-2 SEQUENCES IN ACTIN-BASED MOTORSConsensus docking sites1 for actin-based motility, dened by the oligoproline modules in Listeria monocytogenes ActA surface protein and human platelet vasodilator-stimulated phosphoprotein (VASP). Analysis of Handbook of Biochemical Kinetics

ABORTIVE COMPLEXESNonproductive reversible complexes of an enzyme with various substrates and/or products. The International Union of Biochemistry1 distinguishes dead-end complex from abortive complex, and the latter term is regarded

1

Abscissaas a synonym for nonproductive complex. Complexes that fail to undergo further reactions along the catalytic pathway are called dead-end complexes, and the reactions producing them are called dead-end reactions. Some ambiguity exists in the literature regarding the usage of abortive complex. For example, the term has been used to describe the nonproductive complex formed between an enzyme and a competitive inhibitor2 or to describe that inhibition of depolymerases resulting from shifted registration of the substrate within the enzymes set of subsites2,3. Still others have used the term interchangeably with dead-end complexes4. The term abortive complexes formation is treated as a special case of dead-end complexation and is restricted to nonproductive complexes involving the binding of substrate(s) and/or product(s) to one or more enzyme forms. Thus, nonproductive complexes that culminate in substrate inhibition are abortive complexes. For a discussion concerning formation of EB and EP complexes in rapidequilibrium ordered Bi Bi reactions, see the section on the Frieden Dilemma. Enzyme-substrate-product complexes that often form with multisubstrate enzymes are also abortive complexes. Early product inhibition studies of Aerobacter aerogenes ribitol dehydrogenase5 demonstrated the formation of the E-NAD -D-ribulose and E-NADH-D-ribitol complexes. In the lactate dehydrogenase reaction, the E-NADH-lactate and E-NAD -pyruvate complexes are stable6, and determination of the Kd values indicates that the ENAD -pyruvate ternary complex is physiologically relevant7. Abortive complexes have been reported for a wide variety of enzymes. Isotope exchange at equilibrium is used to identify the E-NADH-malate abortive with bovine heart malate dehydrogenase7. Creatine kinase forms an E-MgADP-creatine complex8. Inhibition at high substrate-product concentrations may arise from factors other than abortive complexes; for example, the inhibition observed in an equilibrium exchange experiment may be related to high ionic strength of reaction solutions9. Wong and Hanes10 pointed out that equilibrium exchange studies can be useful in detecting the presence of abortive species. Although abortive complexes can complicate exchange kinetic behavior, the Wedler-Boyer protocol11 minimizes the inuence of abortives on equilibrium exchange studies. 2 Different abortives may be formed with alternative products or substrates. Such procedures can be useful in helping to distinguish Theorell-Chance mechanisms from ordered systems with abortive complexes12. In the case of lactate dehydrogenase, the E-pyruvate-NAD and Elactate-NADH abortive complexes may play a regulatory roles in aerobic versus anaerobic metabolism. Computer simulations13 also point to the regulatory potential of these non-productive complexes. See Deadend Complexes; Inhibition; Nonproductive Complexes; Product Inhibition; Substrate Inhibition; Isotope Trapping; Isotope Exchange at Equilibrium; Enzyme Regulation1

International Union of Biochemistry (1982) Eur. J. Biochem. 28, 281. 2 M. Dixon & E. C. Webb (1979) Enzymes, 3rd ed., Academic Press, New York. 3 J. D. Allen (1979) Meth. Enzymol. 64, 248. 4 H. J. Fromm (1975) Initial Rate Enzyme Kinetics, Springer-Verlag, New York. 5 H. J. Fromm & D. R. Nelson (1962) J. Biol. Chem. 231, 215. 6 H. J. Fromm (1963) J. Biol. Chem. 238, 2938. 7 H. Gutfreund, R. Cantwell, C. H. McMurray, R. S. Criddle & G. Hathaway (1968) Biochem. J. 106, 683. 8 E. Silverstein & G. Sulebele (1969) Biochemistry 8, 2543. 9 J. F. Morrison & W. W. Cleland (1966) J. Biol. Chem. 241, 673. 10 J. T.-F. Wong & C. S. Hanes (1964) Nature 203, 492. 11 F. C. Wedler & P. D. Boyer (1972) J. Biol. Chem. 247, 984. 12 C. C. Wratten & W. W. Cleland (1965) Biochemistry 4, 2442. 13 D. L. Purich & H. J. Fromm (1972) Curr. Topics in Cell. Reg. 6, 131. Selected entries from Methods in Enzymology [vol, page(s)]: Formation, 63, 43, 419-424, 432-436; chymotrypsin, 63, 205; isotope exchange, 64, 32, 33, 39-45; isotope trapping, 64, 58; limitation, 63, 432-436; multiple, one-substrate system, 63, 473, 474; pH effects, 63, 205; practical aspects, 63, 477-480; substrate inhibition, 63, 500, 501; two-substrate system, 63, 474-478; in product inhibition studies, 249, 188-189, 193, 199-200, 205; identication of, 249, 188-189, 202, 206, 208-209.

ABSCISSAThe x-coordinate axis for a graph of Cartesian coordinates [x,y] or [x, f (x)] or the x-value for any [x,y] ordered pair. This corresponds to the [Substrate Concentration]axis in v versus [S] plots or the 1/[Substrate Concentration]-axis in so-called double-reciprocal or LineweaverBurk plots.

ABSOLUTE CONFIGURATIONA method for designating the stereoisomeric conguration of a chiral carbon atom within a molecular entity. The designation D was arbitrarily assigned to ( )-glyceraldehyde, and ( )-glyceraldehyde was assigned the label Handbook of Biochemical Kinetics

Absorption CoefcientL. Compounds that can be derived from L-glyceraldehyde

ABSORBANCEA quantitative measure of photon absorption by a molecule, expressed as the log10 of the ratio of the radiant intensity Io of light transmitted through a reference sample to the light I transmitted through the solution [i.e., A log(Io /I )]. Out-moded terms for absorbance such as optical density, extinction, and absorbancy should be abandoned. The International Union of Pure and Applied Chemistry recommends that the denition should now be based on the ratio of the radiant power of incident radiation (Po) to the radiant power of transmitted radiation (P). Thus, log T 1. In solution, Po would refer A log(Po /P) to the radiant power of light transmitted through the reference sample. T is referred to as the transmittance. If natural logarithms are used, the quantity, symbolized by B, is referred to as the Napierian absorbance. Thus, B ln(Po /P). The denition assumes that light reection and light scattering are negligible. If not, the appropriate term for log(Po/P) is attenuance. See Beer-Lambert Law; Absorption Coefcient; Absorption Spectroscopy

without inversion reactions of the chiral center are likewise designated L- (and, their mirror images, D-). X-ray crystallographic studies later showed that D-glyceraldehyde had the conguration shown below1. D- and L-Alanine are also depicted.

The DL-system is in common use with respect to amino acids and sugars, but the Cahn-Ingold-Prelog system (the RS-system) is more systematic and should be used. The literature is replete with reports failing to specify the stereochemistry of certain reactions, and one must often infer the enantiomer. See Conguration; CahnIngold-Prelog System; Corn Rule; Diastereomers; Enantiomers; (R/S)-Convention1

J. M. Bijuoet, A. F. Peerdeman & A. J. van Bommel (1951) Nature 168, 271. 2 W. Klyne & J. Burkingham (1978) Atlas of Stereochemistry, 2nd ed., vol. 2, Oxford Univ. Press, New York. 3 J. Jacques, C. Gros, S. Bourcier, M. J. Brienne & J. Toullec (1977) Absolute Congurations, Georg Thieme Publ., Stuttgart.

ABSORBED DOSE1. The quantity of absorbed energy absorbed per unit mass of a substance, object, or organism in an irradiated medium. Symbolized by D, the SI unit is the gray (Gy; joules per kilogram). The unit rad is also commonly used (1 rad 0.01 Gy). 2. The amount of substance (e.g., pharmaceutical) absorbed by an organism or cell.

ABSOLUTE TEMPERATUREA temperature measured on an absolute temperature scale (i.e., a scale in which zero degrees is equivalent to absolute zero). In the Kelvin scale, the degree unit is the kelvin, abbreviated as K; it does not have the superscript o used to indicate degree as on the Celsius scale. K has the same magnitude as degree Celsius ( C).

ABSORPTION1. The process of being taken up or becoming a part of another body. The unrelated, but often confused term adsorption describes a particular type of surface phenomenon. See also Adsorption. 2. The process of transfer of energy (e.g., from an electromagnetic eld) to a structure or entity (e.g., a molecular entity). 3. The process of transport of a substance into a cell.

ABSOLUTE UNCERTAINTYThe uncertainty in measured values expressed in units of the measurement. For example, a reaction velocity of 10.2 M/min is presumed to be valid to a tenth, and the absolute uncertainty is 0.1 M/min. See Relative Uncertainty

ABSOLUTE ZEROThe temperature at thermal energy of random motion of molecular entities of a system in thermal equilibrium is zero. This temperature is equal to 273.15 C or 459.67 F. Note that even at absolute zero, chemical bonds still retain zero point energy. Handbook of Biochemical Kinetics

ABSORPTION COEFFICIENTThe log-base10 attenuance (or absorbance) (i.e., D or A) divided by the optical pathlength (l ). This coefcient, symbolized by a, is thus equal to l 1log(Po /P). See BeerLambert Law; Absorbance; Molar Absorption Coefcient; Absorption Spectroscopy 3

Absorption Spectroscopy

ABSORPTION SPECTROSCOPYAbsorption spectroscopy is widely used to follow the course of enzyme-catalyzed reactions. Absorbance measurements should be made under conditions that permit use of the Beer-Lambert Law: A log (Io /I) cl

rate experiments. Prior centrifugation or ltration may be needed to reduce light scattering or turbidity.

where A is the absorbance (a dimensionless parameter), I0 and I are the incident radiant intensity and the transmitted radiant intensity, is the molar (base10) absorption coefcient, c is the molar concentration of the absorbing species, and l is the absorption pathlength (i.e., the distance through solution that light must pass). [Note: IUPAC1 now favors A log(Po/P) where Po and P are the incident radiant power and the transmitted radiant power, respectively.]

Figure 2. Design features of a double-beam UV/visible spectrophotometer. Note that rays of light pass through a set of slits as they enter the light-tight monochromator. Rotation of the prism determines the wavelength of dispersed light that passes on to the sample compartment. The chopper-motor rotates a beam-splitter that allows half of the in-coming light to travel to the reference compartment, while reecting the other half of the in-coming light to the sample compartment.

Instruments with double monochromator congurations, or equivalent multi-pass congurations, can greatly reduce stray light (which is any radiation of wavelength other than that of the columnated light beam). Absorbance in the presence of stray light can be expressed as: AFigure 1. Perpendicular disposition of the electric vector E and magnetic vector H of a light wave traveling from its source in the direction of propagation shown by the arrow. Note that electromagnetic radiation interacts with molecules in two ways: (a) in absorption, the energy of a photon is absorbed by an electron (hence, the familiar term electronic absorbance spectrum) when the direction of the electric vector is aligned with the transition dipole of the molecule; (b) in light scatterring, only the direction of propagation is changed, and very little, if any, energy is lost.

log [(Io

Is)/(I

Is )]

where Is is the stray light intensity. The larger the value of Is (relative to Io or I), the greater the error in concentration or rate measurements2.

Practical Considerations. Typical absorption assay methods utilize ultraviolet (UV) or visible (vis) wavelengths. With most spectrophotometers, the measured absorbance should be less than 1.2 to obtain a strictly linear relationship (i.e., to obey the Beer-Lambert Law). Nonlinear A versus c plots can result from micelle formation, sample turbidity, the presence of stray light (see below), bubble formation, stacking of aromatic chromophores, and even the presence of ne cotton strands from tissue used to clean the faces of cuvettes. One is well advised to conrm the linearity of absorbance with respect to product (or substrate) concentration under the exact assay conditions to be employed in 4

Figure 3. Example of the BeerLambert relationship.

The Beer-Lambert relationship is additive (i.e., the absorption of light by one chemical species is unaffected Handbook of Biochemical Kinetics

Absorption Spectroscopyby the presence of other species, irrespective of whether those other species absorb light at the same wavelength). Thus, A ( i ci l). The greater the difference in the molar absorption coefcients between the substrate(s) and the product(s), the larger the change in absorbance with time and the greater the ease in velocity determination. This statement assumes that the two chemical species do not interact with each other. Note that the Beer-Lambert relationship does not require one to monitor a reaction at the wavelength maximum value ( max ) of either the substrate or the product. All other factors being equal, one should chose that wavelength yielding the greatest value. For example, AMP has a max value at 259 nm at a pH value of 7, whereas IMP has a max value of 248.5 nm. Yet, the AMP deaminase reaction is measured best at 265 nm, the wavelength affording the largest change in . Likewise, one can use a wavelength other than 340 nm to assay NADH or NADPH, but one should avoid unnecessary loss of signal-to-noise by working as close to the wavelength yielding maximal absorption.

Figure 5. Ultraviolet spectrum of NAD and NADH. Note that the absorption band centered at 340 nm serves as a valuable way to assay many dehydrogenases as well as other enzymes that form products that can be coupled to NAD reduction or NADH oxidation.

Occasionally, one can increase the by utilizing alternative substrates. For example, 3-acetyl-NAD or thioNAD can often be used with NAD -dependent dehydrogenases. Note however that an alternative substrate may change the kinetic mechanism, as compared to that observed with the naturally occurring substrate. Alternative substrates are of particular value when the normal substrate(s) and product(s) do not efciently absorb UV or visible light. For example, many p-nitroaniline or pnitrophenyl derivatives have proved to be quite useful in enzyme assays because they exhibit intense absorption around 410 nm. Ideally, other components in the reaction mixture should not absorb signicantly at the monitored wavelength. In addition, colored impurities should be removed. For example, commercial imidazole, a commonly used buffer, contains a yellow impurity that can be easily removed upon recrystallization from ethyl acetate. General Principles. Light absorption is quantized. The energy change associated with an electronic transition occurs within the ultraviolet (UV) or visible (vis) spectrum. Visible light absorption corresponds to low-energy electronic transitions, such as those observed with certain transition metal ions or with molecules having conjugated double bonds. The near ultraviolet (200 and 400 nm) corresponds to electronic transitions in molecular entities with smaller conjugated systems (e.g., 1,3,5-hexatriene, ATP, etc.). Isolated double bonds, such as those within peptide bonds, absorb light around 200-210 nm. Dioxygen, carbon dioxide, and water can absorb light of 5

Figure 4. Ultraviolet spectrum of bases found in DNA and RNA.


Absorption Spectroscopywavelength less than 180 nm, and spectroscopy in the farUV typically requires a vacuum, hence the term vacuum UV for low-wavelength light. Sharp absorption bands are typically not observed in UV and visible absorption spectra of liquid samples. This is the consequence of the presence of the vibrational and rotational ne structure that become superimposed on the potential energy surfaces of the electronic transitions. Fine structure in UV/vis absorption spectra can be detected for samples in vapor phase or in nonpolar solvents. The intensity of light absorption is governed by a number of factors that determine the transition probability (i.e., the probability of interaction between the radiant energy and the electronic system). This probability is proportional to the square of the transition moment and is thus related to the electronic charge distribution in the molecular entity. Hence, absorption bands (with 10,000 cm 1 M 1) suggest that the transition is accompanied by a large change in the transition moment. Intensity is also affected by the polarity of the excited state and the target area of the absorbing system. Transitions associated with UV-visible absorption spectroscopy consist of an electron in a lled molecular orbital being excited to the next higher energy orbital (an antibonding orbital). Although many exceptions are known, the relative * n * transition energies roughly are: n * *. * Transitions. These transitions typically occur between 120 and 220 nm (i.e., in the far-UV). The max value for typical * transitions of carbon-carbon or carbon-hydrogen bonds is usually around 150 nm. For example, the max value for ethane is 135 nm. n * Transitions. These transitions typically occur at wavelengths greater than that needed for * U * transitions involving UO transitions. Roughly, n occur with wavelengths at about 185 nm, with UN U U at about 195 nm, and with carbonyls at about and US values) of 190 nm. Examples of max values (and * transitions include: water ( max 167 nm with n 183 nm with 500), 7000), methanol ( max acetone ( max 188 nm with 1860), and methyl iodide ( max 259 nm with 400). n * Transitions. These are forbidden transitions according to symmetry rules, but molecular vibrations 6 allow these transitions to occur, albeit with low intensities. Nonbonding electrons of carbonyl groups will often have n * transition max values of around 300 nm. 279 nm, Some examples include acetone ( max 15), acetophenone (319 nm, 50), thiourea (a shoulder at 291 nm, 71), and acetic acid (204 nm, 41). The n * transition can also be detected in optical rotatory dispersion measurements. Moreover, n * transitions often exhibit a blue shift in polar solvents or environments. * Transitions. Typically occurring at wavelengths in the near-UV, transitions of this type are the most commonly utilized spectral signals in kinetic and structural studies.1 2

IUPAC (1988) Pure and Appl. Chem. 60, 1055. R. D. Allison & D. L. Purich (1979) Meth. Enzymol. 63, 3. 3 C. R. Cantor & P. R. Schimmel (1980) Biophysical Chemistry, part II, pp. 344-408, Freeman, San Francisco. 4 R. P. Bauman (1962) Absorption Spectroscopy, Wiley, New York. 5 H. H. Jaffe & M. Orchin (1962) Theory and Application of Ultraviolet Spectroscopy, Wiley, New York. Selected entries from Methods in Enzymology [vol, page(s)]: Absorption Spectrophotometer: Application, 24, 15-25; baseline compensation, 24, 8-10; computerized, 24, 19-25; light scattering, 24, 13-15; monochromator, 24, 4; photometer, 24, 5-8; recorder, 24, 8; sample compartment, 24, 5; single-beam, 24, 3-4; spectral characteristics, 24, 10-12; split-beam, 24, 3; stray light, 24, 12-13. Optical Spectroscopy: General principles and overview, 246, 13; absorption and circular dichroism spectroscopy of nucleic acid duplexes and triplexes, 246, 19; circular dichroism, 246, 34; bioinorganic spectroscopy, 246, 71; magnetic circular dichroism, 246, 110; low-temperature spectroscopy, 246, 131; rapid-scanning ultraviolet/visible spectroscopy applied in stopped-ow studies, 246, 168; transient absorption spectroscopy in the study of processes and dynamics in biology, 246, 201; hole burning spectroscopy and physics of proteins, 246, 226; ultraviolet/visible spectroelectrochemistry of redox proteins, 246, 701; diode array detection in liquid chromatography, 246, 749. Nanosecond Absorption Spectroscopy: Absorption apparatus, 226, 131; apparatus, 226, 152; detectors, 226, 126; detector systems, 226, 125; excitation source, 226, 121; global analysis, 226, 146, 155; heme proteins, 226, 142; kinetic applications, 226, 134; monochromators/spectrographs, 226, 125; multiphoton effects, 226, 141; nanosecond time-resolved recombination, 226, 141; overview, 226, 119, 147; probe source, 226, 124; quantum yields, 226, 139; rhodopsin, 226, 158; sample holders, 226, 133; singular value decomposition, 226, 146, 155; spectral dynamics, 226, 136; time delay generators, 226, 130. Time-Resolved Absorption Spectroscopy: Advantages, 232, 389; applications, 232, 387-388; detectors, 232, 387, 392-393, 399; hemoglobin data analysis, 232, 401-415; kinetic analyses, 232, 390; photoselection effects, 232, 390-391; kinetic intermediates and,


Acetate Kinase232, 389; spectrometer for, 232, 392-401; performance characteristics, 232, 389-390.

the accepted true value for a given quantity. See also Precision

ABSORPTIVITYA parameter in spectroscopy and photochemistry, equal to the absorptance (1 (P/P0 )) divided by the optical pathlength (l) of the sample containing the absorbing agent. Thus, it equals (1 (P/P0 ))/l where P0 is the radiant power of light being transmitted through a reference sample, and P is the radiant power being transmitted through the solution. The Commission on Photochemistry does not recommend the use of this term. See Absorbance; Absorption Coefcient; Beer-Lambert Law; Absorption Spectroscopy

ACETALDEHYDE DEHYDROGENASE (ACETYLATING)This enzyme [EC 1.2.1.10] catalyzes the oxidation of acetaldehyde in the presence of NAD and coenzyme A to form acetyl-CoA NADH H . Other aldehyde substrates include glycolaldehyde, propanal, and butanal.E. R. Stadtman & R. M. Burton (1955) Meth. Enzymol. 1, 222 and 518. F. B. Rudolph, D. L. Purich & H. J. Fromm (1968) J. Biol. Chem. 243, 5539.

ABSTRACTION REACTIONAny chemical process in which one reactant removes an atom (neutral or charged) from the other reacting entity. An example is the generation of a free radical by the action of an initiator on another molecule. If abstraction takes place at a chiral carbon, racemization is almost always observed in nonenzymic processes. On the other hand, enzymes frequently abstract and reattach atoms or groups of atoms in a fashion that maintains stereochemistry.

2-(ACETAMIDOMETHYLENE)SUCCINATE HYDROLASEThis enzyme [EC 3.5.1.29] catalyzes the hydrolysis of 2(acetamidomethylene)-succinate to yield acetate, succinate semialdehyde, carbon dioxide, and ammonia.R. W. Burg (1970) Meth. Enzymol. 18(A), 634.

ACETATE KINASEThis phosphotransferase [EC 2.7.2.1] catalyzes the thermodynamically favored phosphorylation of ADP to form [ATP][acetate]/ [acetyl phosphate] ATP (i.e., Keq [ADP] 3000). GDP is also an effective phosphoryl group acceptor. This enzyme is easily cold-denatured, and one must use glycerol to maintain full catalytic activity. Initial kinetic evidence, as well as borohydride reduction experiments, suggested the formation of an enzymebound acyl-phosphate intermediate, but later kinetic and stereochemical1 data indicate that the kinetic mechanism is sequential and that there is direct in-line phosphoryl transfer. Incidental generation of a metaphosphate anion during catalysis may explain the formation of an enzymebound acyl-phosphate. Acetate kinase is ideally suited for the regeneration of ATP or GTP from ADP or GDP, respectively.1

ACCELERATIONIn physics, the time rate of change of motional velocity resulting from changes in a bodys speed and/or direction. In biochemistry, acceleration refers to an increased rate of a chemical reaction in the presence of an enzyme or other catalyst. See Catalytic Rate Enhancement; Catalytic Prociency; Efciency Function

ACCRETIONSolute or particulate accumulation onto an aggregated phase (or solid state) that grows together by the addition of material at the periphery. Both cohesive and adhesive forces are thought to be driving forces in accretion. Sea shells and kidney stones are also known to form as layers of crystallites and amorphous components by accretion of external substances.

P. A. Frey (1992) The Enzymes 20, 160.

ACCURACYThe closeness or proximity of a measured value to the true value for a quantity being measured. Unless the magnitude of a quantity is specied by a formal SI denition, one typically uses reference standards to establish Handbook of Biochemical Kinetics

Selected entries from Methods in Enzymology [vol, page(s)]: Acetate assay with, 3, 269; activation, 44, 889; activity assay, 44, 893, 894; alternative substrates, 87, 11; bridge-to-nonbridge transfer, 87, 19-20, 226, 232; chiral phosphoryl-ATP, 87, 211, 258, 300; cold denaturation, 63, 9; cysteine residues, 44, 887-889; equilibrium constant, 63, 5; exchange properties, 64, 9, 39, 87, 5, 18, 656; hydroxylaminolysis, 87, 18; immobilization, 44, 891, 892; inhibitor, 63, 398; initial rate kinetics, 87, 18; metal-ion bind-

7

Acetate Kinaseing, 63, 275-278; metaphosphate, 87, 12, 20; metaphosphate synthesis and, 6, 262-263; nucleoside diphosphate kinase activity; 63, 8; phosphorylation potential, 55, 237; pH stability prole, 87, 18; promoting microtubule assembly, 85, 417-419; purine nucleoside diphosphate kinase activity, 63, 8; regenerating GTP from GDP, 85, 417; ribulose-5-phosphate 4-epimerase and, 5, 253254; Veillonella alcalescens acetate kinase [ATP formation assay, 71, 312; hydroxamate assay, 71, 311; properties, 71, 315; stability to heat, 71, 313; stimulation by succinate, 71, 316; substrate specicity, 71, 316]; xylulose-5-phosphate 3-epimerase and, 5, 250-251; xylulose-5-phosphate phosphoketolase and, 5, 26; purine inhibitor, 63, 398; metal-ion binding, 63, 275-278; phosphorothioates, 87, 200, 205, 226, 232, 258; from Bacillus stearothermophilus; assay, 90, 179; properties, 90, 183; purication, 90, 180; in acetyl phosphate and acetyl-CoA determination, 122, 44; and hexokinase, in glucose 6-phosphate production, 136, 52; dihydroxyacetone phosphate synthesis with, 136, 277; glucose 6phosphate synthesis with, 136, 279; sn-glycerol 3-phosphate synthesis with, 136, 276; in pyruvic acid phosphoroclastic system, 243, 96, 99. J. S. Holt, S. B. Powles & J. A. M. Holtum (1993) Ann. Rev. Plant Physiol. Plant Mol. Biol. 44, 203. R. Kluger (1992) The Enzymes, 3rd ed., 20, 271. J. H. Jackson (1988) Meth. Enzymol. 166, 230.

ACETYLCHOLINESTERASEThis enzyme [EC 3.1.1.7], also known as true cholinesterase, choline esterase I, and cholinesterase, catalyzes the hydrolysis of acetylcholine to produce choline and acetate. The enzyme will also act on a number of acetate esters as well as catalyze some transacetylations.D. M. Quinn & S. R. Feaster (1998) Comprehensive Biological Catalysis: A Mechanistic Reference 1, 455. H. Okuda (1991) A Study of Enzymes 2, 563. T. L. Rosenberry (1975) Adv. Enzymol. 43, 103. H. C. Froede & I. B. Wilson (1971) The Enzymes, 3rd ed., 5, 87. L. T. Potter (1971) Meth. Enzymol. 17(B), 778. Selected entries from Methods in Enzymology [vol, page(s)]: Acetylthiocholine as substrate, 251, 101-102; assay by ESR, 251, 102-105; inhibitors, 251, 103; modication by symmetrical disulde radical, 251, 100; thioester substrate, 248, 16; transition state and multisubstrate analogues, 249, 305; enzyme receptor, similarity to collagen, 245, 3.

ACETATE KINASE (PYROPHOSPHATE)This enzyme [EC 2.7.2.12] converts acetate and pyrophosphate to form acetyl phosphate and orthophosphate.H. G. Wood, W. E. OBrien & G. Michaels (1977) Adv. Enzymol. 45, 8555.

ACETYL-CoA (or, ACETYL COENZYME A)

ACETAZOLAMIDEA diuretic agent (5-acetamido-1,3,4-thiadiazole-2-sulfonamide) that acts as a potent noncompetitive inhibitor (Ki 10 8 M) of carbonic anhydrase.

ACETOACETATE DECARBOXYLASEThis enzyme [EC 4.1.1.4] catalyzes the decarboxylation of acetoacetate to form acetone and carbon dioxide.M. H. OLeary (1992) The Enzymes, 3rd ed., 20, 235. D. J. Creighton & N. S. R. K. Murthy (1990) The Enzymes, 3rd ed., 19, 323. D. S. Sigman & G. Mooser (1975) Ann. Rev. Biochem. 44, 889. I. Fridovich (1972) The Enzymes, 3rd ed., 6, 255.

ACETOLACTATE SYNTHASEThis enzyme [EC 4.1.3.18] catalyzes the reversible carboxylation of 2-acetolactate with carbon dioxide to form two pyruvate ions. Thiamin pyrophosphate is a required cofactor.B. A. Palfey & V. Massey (1998) Comprehensive Biological Catalysis: A Mechanistic Reference 3, 83. R. L. Schowen (1998) Comprehensive Biological Catalysis: A Mechanistic Reference 2, 217. J. V. Schloss & M. S. Hixon (1998) Comprehensive Biological Catalysis: A Mechanistic Reference 2, 43. A. Schellenberger, G. Hubner & H. Neef (1997) Meth. Enzymol. 279, 131.

As the principal thiolester of intermediary metabolism, acetyl coenzyme A is involved in two-carbon biosynthetic and degradative steps. An essential component is the vitamin pantithenic acid, which provides the sulfur atom for the thiolester formation.Selected entries from Methods in Enzymology [vol, page(s)]: Assay, 1, 611; 3, 935-938; 63, 33; separation by HPLC, 72, 45; extraction from tissues, 13, 439; formation of, 1, 486, 518, 585; 5, 466; free energy of hydrolysis, 1, 694; substrate for the following enzymes [acetyl-coenzyme A acyl carrier protein transacylase, 14, 50; acetyl-coenzyme A carboxylase, 14, 3, 9; acetyl-coenzyme A synthetase, 13, 375; N-acetyltransferase, 17B, 805; aminoacetone

8


N-Acetylgalactosaminide Sialyltransferasesynthase, 17B, 585; carnitine acetyltransferase, 13, 387-389; 14, 613; choline acetyltransferase, 17B, 780, 788, 798; citrate synthase, 13, 3, 4, 8, 9, 11, 12, 15-16, 19-20, 22, 25; 14, 617; fatty acid synthase, 14, 17, 22, 33, 40.

ACETYL-CoA SYNTHETASEThis enzyme [EC 6.2.1.1], also referred to as acetateCoA ligase or acetate thiokinase, catalyzes the reaction of acetate, coenzyme A, and ATP to form acetyl-CoA, AMP, and pyrophosphate. The enzyme will also utilize propanoate and propenoate as substrates.L. A. Kleczkowski (1994) Ann. Rev. Plant Physiol. Plant Mol. Biol. 45, 339. J. C. Londesborough & L. T. Webster, Jr. (1974) The Enzymes, 3rd ed., 10, 469. Selected entries from Methods in Enzymology [vol, page(s)]: Adenosine 5 -O-(1-thiotriphosphate), 87, 224, 230-231; bridgenonbridge oxygens, 87, 251-253; kinetics, 87, 355; mechanism, 87, 251, 355; NMR, 87, 251-253; stereochemistry 87, 206, 212, 224, 230-233, 251-253.

ACETYL-CoA C-ACETYLTRANSFERASE (or, THIOLASE)This enzyme [EC 2.3.1.9], also known as thiolase, transfers an acetyl group from one acetyl-CoA molecule to another to form free coenzyme A and acetoacetyl-CoA.D. J. Creighton & N. S. R. K. Murthy (1990) The Enzymes, 3rd ed., 19, 323. U. Gehring & F. Lynen (1972) The Enzymes, 3rd ed., 7, 391.

ACETYL-CoA C-ACYLTRANSFERASEThis enzyme [EC 2.3.1.16], also known as 3-ketoacylCoA thiolase, transfers an acyl group from an acyl-CoA to acetyl-CoA to form free coenzyme A and 3-oxoacyl-CoA.J. V. Schloss & M. S. Hixon (1998) Comprehensive Biological Catalysis: A Mechanistic Reference 2, 43.

ACETYLENE MONOCARBOXYLATE HYDRATASEThis enzyme [EC 4.2.1.71] adds water to propynoate to form 3-hydroxypropenoate. The enzyme will also act on 3-butynoate to form acetoacetate.J. V. Schloss & M. S. Hixon (1998) Comprehensive Biological Catalysis: A Mechanistic Reference 2, 43.

ACETYL-CoA:ACP TRANSACYLASEThis enzyme [EC 2.3.1.38], also referred to as acetylCoA:[acyl-carrier protein] S-acetyltransferase, transfers an acetyl group from one acetyl-CoA to an acyl-carrierprotein (ACP) to form free coenzyme A and the acetyl[acyl-carrier-protein]. See also Fatty Acid SynthaseS. J. Wakil & J. K. Stoops (1983) The Enzymes, 3rd ed.,16, 3. P. R. Vagelos (1973) The Enzymes, 3rd ed., 8, 155. A. W. Alberts, P. W. Majerus & P. R. Vagelos (1969) Meth. Enzymol. 14, 50.

N-ACETYLGALACTOSAMINE-4-SULFATE SULFATASEThis enzyme [EC 3.1.6.12] acts on 4-sulfate groups of the N-acetylgalactosamine 4-sulfate moieties in chondroitin sulfate and dermatan sulfate.H. Kresse & J. Glossl (1987) Adv. Enzymol. 60, 217.

N-ACETYLGALACTOSAMINE-6-SULFATE SULFATASEThis enzyme [EC 3.1.6.4] acts on 6-sulfate groups of the N-acetylgalactosamine 6-sulfate moieties in chondroitin sulfate and the galactose 6-sulfate groups in keratan sulfate.H. Kresse & J. Glossl (1987) Adv. Enzymol. 60, 217.

ACETYL-CoA CARBOXYLASEThis enzyme [EC 6.4.1.2] catalyzes the reaction of acetylCoA, bicarbonate, and ATP to form malonyl-CoA, orthophosphate, and ADP. The plant enzyme will also act on propionyl-CoA and butanoyl-CoA. The enzyme will also catalyze certain transcarboxylations and it requires biotin as a cofactor.J. N. Earnhardt & D. N. Silverman (1998) Comprehensive Biological Catalysis: A Mechanistic Reference 1, 495. J. V. Schloss & M. S. Hixon (1998) Comprehensive Biological Catalysis: A Mechanistic Reference 2, 43. K.-H. Kim (1997) Ann. Rev. Nutr. 17, 77. S. B. Ohlrogge & J. G. Jaworski (1997) Ann. Rev. Plant Physiol. Plant Mol. Biol. 48, 109. R. W. Brownsey & R. M. Denton (1987) The Enzymes, 3rd ed., 18, 123. K. Bloch (1977) Adv. Enzymol. 45, 1. A. W. Alberts & P. R. Vagelos (1972) The Enzymes, 3rd ed., 6, 37.

N-ACETYLGALACTOSAMINIDE SIALYLTRANSFERASEThis enzyme [EC 2.4.99.3] catalyzes the reaction of a glycano-1,3-(N-acetylgalactosaminyl)-glycoprotein and CMP-N-acetylneuraminate to produce CMP and the glycano-(2,6- -N-acetylneuraminyl)-(N-acetylgalactosaminyl)-glycoprotein.T. A. Beyer, J. E. Sadler, J. I. Rearick, J. C. Paulson & R. L. Hill (1981) Adv. Enzymol. 52, 23.


9

N-Acetylglucosamine Kinase

N-ACETYLGLUCOSAMINE KINASEThis enzyme [EC 2.7.1.59] catalyzes the phosphorylation by ATP of N-acetylglucosamine to generate ADP and N-acetylglucosamine 6-phosphate. The bacterial enzyme is also reported to act on glucose as well.S. S. Barkulis (1966) Meth. Enzymol. 9, 415.

S. G. Powers-Lee (1985) Meth. Enzymol. 113, 27. T. Sonoda & M. Tatibana (1983) J. Biol. Chem. 258, 9839. H. J. Vogel & R. H. Vogel (1974) Adv. Enzymol. 40, 65.

N-ACETYL- -GLUTAMYL-PHOSPHATE REDUCTASEThis enzyme [EC 1.2.1.38], also known as N-acetylglutamate semialdehyde dehydrogenase and NAGSA dehydrogenase, catalyzes the reaction of N-acetylglutamate 5-semialdehyde with NADP and phosphate to generate N-acetyl-5-glutamyl phosphate and NADPH.H. J. Vogel & R. H. Vogel (1974) Adv. Enzymol. 40, 65.

N-ACETYLGLUCOSAMINE-6-PHOSPHATE 2-EPIMERASEThis enzyme catalyzes the epimerization at the 2-position of N-acetylglucosamine 6-phosphate. See also N-Acylglucosamine-6-phosphate 2-EpimeraseM. E. Tanner & G. L. Kenyon (1998) Comprehensive Biological Catalysis: A Mechanistic Reference 2, 7.

-N-ACETYLHEXOSAMINIDASEThis enzyme [EC 3.2.1.52], also referred to as -hexosaminidase and N-acetyl- -glucosaminidase, catalyzes the hydrolysis of terminal nonreducing N-acetylhexosamine residues in N-acetyl- -hexosaminides. N-Acetylglucosides and N-acetylgalactosides are substrates.H. Kresse & J. Glossl (1987) Adv. Enzymol. 60, 217. H. M. Flowers & N. Sharon (1979) Adv. Enzymol. 48, 29.

N-ACETYLGLUCOSAMINE-6-SULFATE SULFATASEThis enzyme [EC 3.1.6.14] catalyzes the hydrolysis of the 6-sulfate moieties of the N-acetylglucosamine 6-sulfate subunits of heparan sulfate and keratan sulfate. It has been suggested that this enzyme might be identical to N-sulfoglucosamine-6-sulfatase.H. Kresse & J. Glossl (1987) Adv. Enzymol. 60, 217.

O -ACETYLHOMOSERINE (THIOL)-LYASEThis enzyme [EC 4.2.99.10], also referred to as O-acetylhomoserine sulfhydrylase, catalyzes the reaction of Oacetylhomoserine with methanethiol to generate methionine and acetate. The enzyme can also act on other thiols or H2S, producing homocysteine or thioethers. The enzyme isolated from bakers yeast will also catalyze the reaction exhibited by O-acetylserine (thiol)-lyase [EC 4.2.99.8], albeit more slowly.S. Yamagata (1987) Meth. Enzymol. 143, 465. I. Shiio & H. Ozaki (1987) Meth. Enzymol. 143, 470.

-N-ACETYLGLUCOSAMINIDASEThis enzyme [EC 3.2.1.50] catalyzes hydrolysis of terminal nonreducing N-acetylglucosamine residues in N-acetyl- -glucosaminides.H. Kresse & J. Glossl (1987) Adv. Enzymol. 60, 217.

-N-ACETYLGLUCOSAMINIDASEThis enzyme, reportedly catalyzing the hydrolysis of terminal, nonreducing N-acetyl- -glucosamine moieties in chitobiose and higher analogs, is now a deleted EC entry [EC 3.2.1.30].P. M. Dey & E. del Campillo (1984) Adv. Enzymol. 56, 141.

N-ACETYLNEURAMINATE LYASEThis enzyme [EC 4.1.3.3], also known as N-acetylneuraminate aldolase, will convert N-acetylneuraminate to N-acetylmannosamine and pyruvate. The enzyme will also act on N-glycoloylneuraminate and on O-acetylated sialic acids, other than O4-acetylated derivatives.K. N. Allen (1998) Comprehensive Biological Catalysis: A Mechanistic Reference 2, 135. W. A. Wood (1972) The Enzymes, 3rd ed., 7, 281.

N-ACETYLGLUTAMATE SYNTHASEThis enzyme [EC 2.3.1.1], also referred to as aminoacid N-acetyltransferase and acetyl-CoA : glutamate Nacetyltransferase, catalyzes the reaction of acetyl-CoA with glutamate to form coenzyme A and N-acetylglutamate. The enzyme will also acts on aspartate and, more slowly, with some other amino acids. The mammalian enzyme is activated by L-arginine. See also Glutamate Acetyltransferase 10

N 2-ACETYLORNITHINE AMINOTRANSFERASEThis enzyme [EC 2.6.1.11] catalyzes the pyridoxal-phosphate-dependent reaction of 2-acetylornithine with Handbook of Biochemical Kinetics

Acid Catalysisketoglutarate to produce N-acetylglutamate 5-semialdehyde and glutamate.H. J. Vogel & R. H. Vogel (1974) Adv. Enzymol. 40, 65. A. E. Braunstein (1973) The Enzymes, 3rd ed., 9, 379. H. J. Vogel & E. E. Jones (1970) Meth. Enzymol. 17(A), 260.

pKa Values for Selected Substances at 25 CSubstance Hydroxyproline (pK1 ) Proline (pK1 ) Aspartic acid (pK1 ) Threonine (pK1 ) Phosphoric acid (pK1 ) Serine (pK1 ) Chloroacetic acid Glycine (pK1 ) Alanine (pK1 ) Formic acid Lactic acid Aspartic acid (pK2 ) Succinic acid (pK1 ) Acetic acid Propionic acid Succinic acid (pK2 ) Carbonic acid (apparent pK1 ) Glucose 1-phosphate (pK2 ) Phosphoric acid (pK2 ) Threonine (pK2 ) Serine (pK2 ) Ammonium ion Ethanolammonium ion Hydroxyproline (pK2 ) Glycine (pK2 ) Trimethylammonium ion Alanine (pK2 ) Aspartic acid (pK3 ) Carbonic acid (pK2 ) Methylammonium ion Proline (pK2 ) Dimethylammonium ion Water (pKw )1

N 2-ACETYLORNITHINE DEACETYLASEThis enzyme [EC 3.5.1.16], also known as acetylornithinase and N-acetylornithinase, catalyzes the reaction of water with N 2-acetylornithine to produce acetate and ornithine. The enzyme also catalyzes the hydrolysis of N-acetylmethionine.H. J. Vogel & R. H. Vogel (1974) Adv. Enzymol. 40, 65.

O-ACETYLSERINE (THIOL)-LYASEThis enzyme [EC 4.2.99.8], also known as cysteine synthase and O-acetylserine sulfhydrylase, catalyzes the pyridoxal-phosphate-dependent reaction of H2S with O 3acetylserine to produce cysteine and acetate. Some alkyl thiols, cyanide, pyrazole, and some other heterocyclic compounds can also act as acceptors.T. Nagasawa & H. Yamada (1987) Meth. Enzymol. 143, 474. A. E. Martell (1982) Adv. Enzymol. 53, 163. L. Davis & D. E. Metzler (1972) The Enzymes, 3rd ed., 7, 33.

pK a 1.82 1.97 2.00 2.10 2.15 2.19 2.28 2.35 2.35 3.75 3.86 3.91 4.21 4.76 4.87 5.63 6.35 6.50 7.20 9.10 9.21 9.25 9.50 9.66 9.78 9.79 9.87 10.00 10.33 10.62 10.64 10.77 13.997

J. T. Edsall & J. Wyman (1958) Biophysical Chemistry, pp. 452, Academic Press, New York.

ACID CATALYSIS

ACHIRALThe absence of chirality. Achiral molecules have an internal plane or point of symmetry. A molecular conguration is said to be achiral when it is superimposable on its mirror image. See Chirality

ACIDA substance that liberates protons as a consequence of its dissolution or dissociation. See Brnsted Theory; Lewis Acid; Lewis Base

ACID-BASE EQUILIBRIUM CONSTANTSThe following table contains pK values for a selected group of biochemically important substances. The associated thermodynamic parameters and the original literature references were presented by Edsall and Wyman1. Handbook of Biochemical KineticsAny process for which the rate of reaction is accelerated through the participation of an acid as a catalyst. See General Acid Catalysis; Specic Acid Catalysis

11

Acidity

ACIDITY1. The tendency for a Brnsted acid to act as a proton donor, expressed in terms of the compounds dissociation constant in water. 2. The term also refers to the tendency to form a Lewis adduct, as measured by a dissociation constant. With reference to a solvent, this term is usually restricted to Brnsted acids. If the solvent is water, the pH value of the solution is a good measure of the proton-donating ability of the solvent, provided that the concentration of the solute is not too high. For concentrated solutions or for mixtures of solvents, the acidity of the solvent is best indicated by use of an acidity function. See Degree of Dissociation; Henderson-Hasselbalch Equation; AcidBase Equilibrium Constants; Brnsted Theory; Lewis Acid; Acidity Function; Leveling Effect

activity coefcients of the indicator. Ho and ho are related log ho . For dilute solutions, Ho pH. This by Ho is the situation for most biochemical reactions. The subscript is a reference to the net charge on the base. Hence, H is the corresponding acidity function for mononegatively charged bases in equilibrium with neutral acids. The Hammett acidity function only applies to acidic solvents having a high dielectric constant, further requiring that the f I /f HI ratio be independent of the nature of the indicator. Thus, the Hammett acidity function is applicable for uncharged indicator bases that are aniline derivatives. As mentioned above, other acidity functions have been suggested. No single formulation has been developed that satises different solvent systems or types of bases. Bunnett and Olsen68 used a different approach and derived the equation: log([SH ]/[S]) Ho (Ho log[H ]) pKSH

ACIDITY FUNCTIONA thermodynamic measure of the proton-donating or proton-accepting ability of a solvent system (or closely related thermodynamic property such as the ability of a solvent system to form Lewis adducts)13. There are many types of acidity scales: depending on the nature of the indicator, of conjugate bases with a 1 charge, of acids that form stable carbocations, etc. Perhaps the best known acidity function is the Hammett acidity function, Ho , which is used for concentrated acidic solvents having a high dielectric constant4,5. For any solvent (including a mixture of solvents in which the relative proportions are specied), Ho is dened to be log([BH ]/[B]). The value of Ho is measured pKBHW using a weak indicator base whose pK value in water is known. Two common indicators are the aniline derivatives, o-nitroanilinium ion and 2,4-nitroanilinium ion, having pKBHW values of 0.29 and 4.53, respectively, in water. The [BH ]/[B] ratio for one indicator is measured, usually spectrophotometrically, in the given solvent. Knowledge of a substances pK in water (i.e., pKBHW ) allows the investigator to calculate Ho . Once Ho is known, pKa values can be determined for any other acid-base pair.

in which S is a base that can be protonated by an acidic Ho versus Ho solvent. Plotting log([SH ]/[S]) log[H ] will result in a reasonably linear line having a slope of . The method uses a linear free energy relationship to address the problems of dening basicity for weak organic bases in solutions of moderate concentrations of mineral acids. The value and sign of is a measure of the response of the acid-base equilibria to changes in acid concentration. An analogous equation for kinetic data is log k Ho (Ho log[H ]) log k2o

where k is the pseudo-rst-order rate constant for a reaction in acidic solution and k2o is the corresponding constant at innite dilution in water. Here is suggested to represent the response of the chemical reaction rate to changes in the acid concentration. Attempts have been made to apply this method to basic media. MoreOFerrell9 suggested that the slope may be related semiquantitatively to the structure of the transition state. A related treatment of the acidity function considers changes in acidity of the solvent on an acid-base equilibria10. With this procedure, an equilibrium is chosen as reference, having an equilibrium constant K o for the Handbook of Biochemical Kinetics

The value of ho is dened as aH fI /f HI where aH is the chemical activity of the proton, and f I and f HI are the 12

Acrosinreference reaction in a reference solvent and K for the reference reaction in the particular medium under study. The reaction under study has the corresponding equilibria of Ko (in the reference solvent) and K (in the particular medium). Thus, log (K /Ko ) m* log (K/Ko ) orthophosphate. The enzyme, which has a wide specicity, will also catalyze transphosphorylations.M. Cohn (1982) Ann. Rev. Biophys. Bioeng. 11, 23. V. P. Hollander (1971) The Enzymes, 3rd ed., 4, 449.

ACONITASEThis [4Fe-4S] cluster-containing enzyme [EC 4.2.1.3], also known as citrate hydro-lyase and aconitate hydratase, will act on citrate to generate cis-aconitate ((Z)prop-1-ene 1,2,3-tricarboxylate) and water. The enzyme will also catalyze the conversion of isocitrate into cisaconitate.V. E. Anderson (1998) Comprehensive Biological Catalysis: A Mechanistic Reference 2, 115. B. G. Fox (1998) Comprehensive Biological Catalysis: A Mechanistic Reference 3, 261. J. V. Schloss & M. S. Hixon (1998) Comprehensive Biological Catalysis: A Mechanistic Reference 2, 43. H. Lauble, M. C. Kennedy, H. Beinert & D. C. Stout (1992)Biochemistry 31, 2735. L. Zheng, M. C. Kennedy, H. Beinert & H. Zalkin (1992) J. Biol. Chem. 267, 7895. J. B. Howard & D. C. Rees (1991) Adv. Protein Chem. 42, 199. P. A. Srere (1975) Adv. Enzymol. 43, 57. J. P. Glusker (1971) The Enzymes, 3rd ed., 5, 413.

where the slope m* corresponds to 1 in the BunnettOlsen treatment. Bunnett11 has also plotted log k Ho vs. log awater where awater is the activity of water and has indicated that the slope of the line, w, suggests certain possibilities for the chemical reaction mechanism in moderately concentrated aqueous acids. For example, if w is between 2.5 and zero, then water does not participate in the formation of the transition state. If w is between 1.2 and 3.3, water participates as a nucleophile in the rate-determining step. Long and Bakule12 have criticized this approach. See also Degree of Dissociation; Henderson-Hasselbalch Equation; Acid-Base Equilibrium Constants; Bunnet-Olsen Equations; Cox-Yeats Treatment1

C. H. Rochester (1970) Acidity Functions, Academic Press, New York. 2 J. March (1985) Advanced Organic Chemistry, Wiley, New York. 3 J. Hine (1962) Physical Organic Chemistry, McGraw-Hill, New York. 4 L. Zucker & L. P. Hammett (1939) J. Amer. Chem. Soc. 61, 2791. 5 L. P. Hammett (1970) Physical Organic Chemistry. McGraw-Hill, New York. 6 J. F. Bunnett & F. P. Olsen (1966) Can. J. Chem. 44, 1899 and 1917. 7 J. F. Bunnett, R. L. McDonald & F. P. Olsen (1974) J. Amer. Chem. Soc. 96, 2855. 8 V. Lucchini, G. Modena, G. Scorrano, R. A. Cox & Y. Yates (1982) J. Am. Chem. Soc. 104, 1958. 9 R. A. More OFerrall (1972) J. Chem. Soc., Perkin Trans. 2, 976. 10 A. Bagno, G. Scorrano & R. A. More OFerrall (1987) Rev. Chem. Interm. 7, 313. 11 J. F. Bunnett (1961) J. Amer. Chem. Soc. 83, 4956. 12 F. A. Long & R. Bakule (1963) J. Amer. Chem. Soc. 85, 2313.

ACONITATE DECARBOXYLASEThis enzyme [EC 4.1.1.6] catalyzes the conversion of cisaconitate to itaconate (or, 2-methylenesuccinate) and carbon dioxide.J. V. Schloss & M. S. Hixon (1998) Comprehensive Biological Catalysis:A Mechanistic Reference 2, 43. R. Bentley (1962) Meth. Enzymol. 5, 593.

ACONITATE

-ISOMERASE

ACID-LABILE SULFIDESThe bridging sulfur atoms in iron-sulfur proteins are often referred to as acid-labile suldes, because treatment of such proteins with acids generates H2S.

This enzyme [EC 5.3.3.7] catalyzes the interconversion of trans-aconitate to cis-aconitate, the reaction reportedly to occur by an allelic rearrangement.D. J. Creighton & N. S. R. K. Murthy (1990) The Enzymes, 3rd ed., 19, 323.

ACROSIN ACID PHOSPHATASEThis enzyme [EC 3.1.3.2], also referred to as acid phosphomonoesterase, phosphomonoesterase, and glycerophosphatase, catalyzes the hydrolysis of an orthophosphoric monoester to generate an alcohol and Handbook of Biochemical Kinetics This enzyme [EC 3.4.21.10] catalyzes the hydrolysis of Arg-Xaa and Lys-Xaa peptide bonds. The enzyme belongs to the peptidase family S1 and is inhibited by naturally occurring trypsin inhibitors.J. S. Bond & P. E. Butler (1987) Ann. Rev. Biochem. 56, 333.

13

Actin Assembly Assays

ACTIN ASSEMBLY ASSAYSThe 43 kDa actin monomer (often termed globular actin or G-actin) polymerizes to form lamentous (or F-) actin, a process that can be measured by a number of biochemical and biophysical techniques1. Actin self-assembly obeys the kinetics of nucleated polymerization2, and the stages of polymerization include nucleation, elongation, and polymer length redistribution. Cooper and Pollard1 have presented the following table indicating the advantages and disadvantages of each of these techniques.

direct assessment of the length of suitably xed and contrast-stained actin laments. (7) DNase inhibition of actin polymerization takes advantage of the observation that DNase I preferentially binds to actin monomers with sufcient afnity to block any polymerization of DNase-actin complex. (8) Pelleting relies on the much higher sedimentation coefcient of actin laments as compared to monomeric actin. (9) Millipore ltration assays allow one to rapidly separate monomeric and polymeric actin using lter disks with 0.45 m pores.

Table I

Methods to Measure Actin PolymerizationMethod 1. Capillary viscometry2. OD232 3. Flow birefringence 4. Fluorescence a. NBD-NEM-actin b. Pyrene-actin 5. Light scattering 6. Electron microscopy 7. DNase inhibition 8. Pelleting 9. Millipore ltration Yes Yes Yes Yes Yes Yes Unknown Moderate High High High High High Moderate No No No Yes No Yes Yes High 0 0 0 0 0 Moderate No No Yes Yes Yes Yes Yes High High High High Moderate Moderate Low 0.5 0.5 1 0.1 0.1 0.2 0.1

Signal [polymer]Yes

Signal : noise ratio HighModerate High

Sensitivity to length YesNo Yes

Shear rate High0 Variable

Native actin YesYes Yes

Expense LowHigh High

Sample size (ml) 0.60.6 1

(1) Capillary viscometry is based on the higher viscosity of F-actin compared to G-actin, and one determines the time needed for a solution to pass through the orice of a glass capillary viscometer. (2) Difference spectroscopy at 232 nm can be utilized to estimate the amount of Factin in a solution, but one must exercise care to correct for light scattering contributions to the apparent optical density change ( OD232). (3) Flow birefringence relies on the alignment of actin laments with the direction of ow imparted by the rotation of one of two concentric cylinders containing the laments in the annular space between the cyclinders. (4) Fluorescence changes in an extrinsic chromophore (NBD or pyrene) covalently attached to actin monomers at cysteine-373 in th

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