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

Dissociation constants in enzyme kinetics KR Dissociation constant for ligand that binds to R-state of allosteric protein KS Equilibrium constant for dissociation of ES complex KT Dissoc

Handbook of Biochemical

Kinetics

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Handbook of Biochemical

Kinetics

Daniel L Purich

R Donald Allison

Department of Biochemistry and Molecular Biology

University of Florida College of Medicine Gainesville, Florida

ACADEMIC PRESS

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 fromthe publisher

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The biotic world is doubtlessly the best known

exam-ple of what Nobelist Murray Gell-Mann has termed

‘‘complex adaptive systems’’—a 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 cell’s 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,

espe-cially 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

investiga-tors 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

inter-actions responsible for homeostasis One approach for

analyzing the mechanics of complex systems is to

deter-mine the chronology of discrete steps within the overall

process—a pursuit called ‘‘kinetics.’’ This strategy allows

an investigator to assess the structural and energetic

de-terminants of transitions from one step to the next By

identifying voids in the time-line, one considers the

possi-bility of other likely intermediates and ultimately

identi-fies all elementary reactions of a mechanism

Kinetics is an analytical approach deeply rooted in

chem-istry and physics, and biochemists have intuitively and

inventively honed the tools of chemists and physicists

for experiments on biological processes Biochemical

ki-netics first began to flourish in enzymology—a field

which has gainfully exploited advances in physical

or-ganic chemistry, structural chemistry, and spectroscopy

in order to dissect the individual steps comprising

en-zyme mechanisms No apology is offered, nor should any

be required, for our strong emphasis on chemical kinetics

and enzyme kinetics Scientists working within these

dis-ciplines have enjoyed unparalleled success in dissectingcomplex multistage processes Mechanisms are tools forassessing current knowledge and for designing betterexperiments As working models, mechanisms offer thevirtues of simplicity, precision, and generativity ‘‘Sim-plicity’’ arises from the symbolic representation of theinteractions among the minimal number of componentsneeded 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’’ resultsfrom the recombining of a model’s constituent elements

to admit new findings, to predict new properties, and tostimulate additional rounds of experiment For chemicaland enzyme kineticists, the goal of this recursive enter-prise is to determine a mechanism (a) that accounts forresponses to changes in each component’s concentration,(b) that explains the detailed time-evolution of all chemi-cal events, (c) that defines the concentration and struc-ture of transient intermediates, (d) that makes sense of

relevant changes in positional properties (i.e.,

conforma-tion, configuraconforma-tion, and/or physical location), and (e)that reconciles the thermodynamics of all reactions stepsand transitions In this respect, the rigor of chemical andenzyme kinetics teaches us all how best to invent newapproaches that appropriately balance theory and exper-iment

The inspiration for this HANDBOOK stemmed from ourshared interest in teaching students about the logical andsystematic investigation of enzyme catalysis and meta-bolic control We began twenty-five years ago with theteaching of graduate-level courses (‘‘Chemical Aspects

of Biological Systems’’; ‘‘Enzyme Kinetics and nism’’) at the University of California Santa Barbara aswell as a course entitled ‘‘Enzyme Kinetics’’ at the Cor-nell University Medical College More recently, we have

taught undergraduate students (‘‘A Survey of

Biochem-istry and Molecular Biology’’) as well as graduate

stu-dents (‘‘Advanced Metabolism’’; ‘‘Physical Biochemistry

and Structural Biology’’; ‘‘Dynamic Processes in the

Mo-lecular 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 notes—an activity

that encouraged us to consider writing what we initially

had envisioned as a short textbook on biochemical

ki-netics

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

termi-nology adequately In 1789, the French chemist Antoine

Lavoisier aptly declared: ‘‘Every branch of physical

sci-ence 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

termi-nology of biochemical kinetics Furthermore, as the

dis-tinction between scientific 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

terminol-ogy Far too much time is wasted when students trace a

finger over many pages of a textbook, only to find a

partial definition 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 definitions

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

respects resembles the ‘‘Micropaedia’’ section of theE CYCLOPAEDIABRITANNICA One loses the seamless orga-nization that can be realized in multichapter expositionsthat systematically develop a series of topics We haveaccordingly attempted to mitigate this problem by in-cluding longer tracts on absorption and fluorescencespectroscopy, biomineralization, chemical kinetics, en-zyme kinetics, Hill and Scatchard treatments, ligandbinding cooperativity, kinetic isotope effects, and proteinpolymerization Likewise, we have extensively insertedcross-references at appropriate locations within manyentries One intrinsic advantage of a mini-encyclopedia,however, is that in subsequent printings we should beable to make corrections and additions, or even deletions

N-of an entire term, without upsetting the overall format

We also felt that readers should be encouraged to consultthe most authoritative sources on particular topics Forthis reason, we developed a collection of over 5000 litera-ture references and, in many cases, our citations creditthe original papers on a given topic We have includedthe names of nearly 1000 enzymes, along with chemicalreactions, EC numbers, and, in many instances, theirbiochemical and catalytic properties The referencescited are not intended to be comprehensive; rather, theyserve to guide the reader to further interesting and help-ful reading on subjects we have discussed Where possi-ble, at least one reference is included to provide informa-tion on assay protocols for the listed enzyme We alsourge readers to use the Wordfinder (included at the back

of the Handbook) to take fullest advantage of the textand reference material The nearly 8000 entries in theWordfinder represent all listed source words as well asother subheadings, keywords, or synonyms Each entry

is immediately followed by the recommended sourceentries, 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 theMETHODS

INENZYMOLOGYfamily of editors for well over two cades The volumes in this series on ‘‘Enzyme Kineticsand Mechanism’’ have become a standard for those inter-ested in biological catalysis As the form of this bookbegan to emerge, we quickly recognized thatMETHODS

de-INENZYMOLOGYcould serve as an additional source forannotations on recommended theories and practices forkinetic studies on a wide range of topics The Handbookcontains nearly 6000 METHODS IN ENZYMOLOGY cita-

tions, and we have indicated the topic, volume, and

be-ginning page for each at the foot of many of the source

words We trust that users of our Handbook will benefit

from improved access to the first 280 volumes ofM

ETH-ODS INENZYMOLOGY

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

toler-ance 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 Fromm’s systematic

‘‘the-ory-of-graphs’’ method as well as W W Cleland’s 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 Huang’s

truly outstanding treatment

The success of ourHANDBOOK OFBIOCHEMICALK

INET-ICScan 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 kinetics—and certainly

not within a first edition We are also certain that, despite

a determined effort to cover the terminology of chemical

and enzyme kinetics, we have still overlooked some portant issues We had also hoped to include additionalkinetic techniques applied in pharmacokinetics, cell biol-ogy, electrophysiology, and metabolic control analysis.Time constraints also prevented our developing mathe-matical sections on Laplace transforms, vector algebra,distribution functions, and especially statistics Eventu-ally, we aspire to create a compact disk version of thisHandbook, appropriately presented as hyperlinked text;that same CD should have room for selected problems/exercises along with step-by-step solutions, as well asdown-loadable programs for kinetic simulation, algo-rithms for symbolic derivation of rate equations, mo-lecular dynamics and related modeling techniques, andtried-and-true statistical methods We also hope that ourreaders will not hesitate to advise us of shortcomings,missed terms, as well as techniques meriting definition,mention, or further explanation We shall be forevergrateful for such guidance

im-We thank our students and colleagues for reading earlierdrafts of our manuscript, and we are especially grateful

to both Shirley Light and Dolores Wright of AcademicPress for their insights, help, and thorough editing of thetext We also thank Academic Press for allowing us toincorporate the numerous annotations to METHODS IN

ENZYMOLOGY

Finally, as first- and second-generation disciples of fessor Herbert J Fromm, we dedicate this book to him,

Pro-in recognition of his germPro-inal and Pro-indelible contributions

to the field of enzyme kinetics and mechanism

Daniel L Purich

R Donald Allison

January, 1999

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& Symbols

Roman Letters and Symbols

A Molecule in the ground state

Acceptor molecule (in fluorescence)A* Molecule in the excited state

A SI symbol for absorbance (unitless)

SI symbol for Helmholtz energy ( J )

SI symbol for the pre-exponentialterm in Arrhenius equation(mol⫺ 1m3)n⫺ 1s⫺ 1

%A Percent absorption of light

(100 ⫺ %T)

A, B, C, Substrate A, B, C,

A, B, C, Coulombic contributions to the

potential energy of interactionMoments of inertia of transition-statecomplex

A ij Amplitude of kinetic decay

Debye-Bi,j Second virial coefficient for the

mutual interactions of species i and j

Bq Becquerel (unit of radioactivity ⫽ 1

disintegration per second)

C or c Molar concentration (M or moles/L)

C SI symbol for heat capacity ( J K⫺ 1)

⌬Cp⬚ Constant pressure standard heat

capacity per mole

C ˆ i or cˆ i Weight concentration of the ith

D SI symbol for debye (unitless)

Donor molecule (in fluorescence)

D SI symbol for translational diffusion

constant (m2s⫺ 1)Spectroscopic energy of dissociation

of a diatomic molecule in the Morseequation

⌬Do Difference in dissociation energies of

products and reactants measured fromzero-point energies

Drot Rotational diffusion constant

d20,w Density extrapolated to 20⬚C, water

Collision diameter

General symbol for enzymeEffector molecule

Abbreviations & Symbols

E⬚ Standard electromotive force

Es Energy of molecule in excited singlet

Momentum distribution function

Number of sites for acceptor onligand (so-called ‘‘ligand valence’’)Oscillator strength

Translational frictional coefficient

f Fractional attainment of isotopic

equilibrium

Frel Fluorescencesample/Fluorescencestandard

ge Degeneracy of the lower state

gu Degeneracy of the upper state

H Henry (unit of self-inductance and

⌬ H ⬚ Standard enthalpy change per mole

⌬‡H ⬚ Standard enthalpy of activation

Hres Magnetic field intensity at which

resonance takes place

Hz Hertz (unit of frequency cycles per

I50or I0.5 Inhibitor yielding 50% inhibition or

0.5 the uninhibited rate

I(␭) Intensity of light at wavelength␭

I(␭)f Intensity of emitted light at

wavelength␭

Abbreviations & Symbols

J Nuclear magnetic resonance coupling

coefficientFlux density (units ⫽ particles area⫺ 1

time⫺ 1)

j Apparent order of a binding reaction

K or Keq Macroscopic equilibrium constant

Ka Acid dissociation constant

KA, KB, Dissociation constant for ligand A, B,

an allosteric protein

Ki Macroscopic inhibition constant

Macroscopic ionization constant

Kia, Kib, Dissociation constants in enzyme

kinetics

KR Dissociation constant for ligand that

binds to R-state of allosteric protein

KS Equilibrium constant for dissociation

of ES complex

KT Dissociation constant for ligand that

binds to T-state of an allostericprotein

Kw The constant equal to the product of

[H⫹] (or, [H3O⫹]) and [OH⫺] in anaqueous solution

K1, K2, K3, Stepwise binding or dissociation

constants for successive attachments

of ligand to an oligomeric receptor

k or kB Boltzmann constant

Microscopic equilibrium constant

kcat Catalytic constant; turnover number

kcat/Km Specificity constant

kd Intrinsic dissociation constant

(reciprocal of ki,j)

kiH, kiD, kiT A rate constant for isotopic isomers

containing H, D, or T

kH/kD Kinetic isotope effect

ki,j Intrinsic association or binding

constant (reciprocal of kd) for

interaction between sites on species i and j

Mn Number average molecular weight

Mr Relative molecular mass

Mw Weight average molecular weight

nHor nHill Hill coefficient

P Generalized symbol for product

P or p Pressure

pKa ⫺log10Ka

pO2 Oxygen partial pressure

Abbreviations & Symbols

(pO2)0.5 Oxygen partial pressure at 0.5

saturation

Q Coulomb (unit of electrostatic charge)

Q Heat absorbed by a defined system

QCO2 Amount CO2released by tissue

Qsyn Synergism quotient

Electric resistanceGross rate of isotopic exchange

SA Partial molal entropy

SA⬘ Unitary part of the partial molal

entropy

⌬‡S⬚ Standard entropy of activation

S1 Singlet state

s Sedimentation coefficient

Equilibrium constant for helix growth

s20,w Sedimentation coefficient corrected to

T1 Longitudinal relaxation time

T2 Transverse relaxation time

Vmor Vmax Maximal velocity

Vm,for Vmax,f Maximal velocity in the forward

Reduced concentration ([F]/KF) forallosteric protein

움H Hill coefficient

웁 Reduced concentration ([I]/KI) for

allosteric protein

Abbreviations & Symbols

⌫ Surface concentration (mol m⫺ 2)

Parameter affecting relaxationamplitude

웂 Reduced concentration ([A]/KA) for

␬ Transmission coefficient for transition

stateInverse screening length

Kinetic decay time

애i Chemical potential of ith species per

␾ Quantum yield (unitless)

␹ Mole fraction of component I

⬍兩兩⬎ Expectation value integral

* Superscript designating radioactive

substanceSuperscript designating excited stateSubscript designating complexconjugate

‡ Superscript for transition state

(⭸x/⭸t)y Partial differential of x with respect to

Abbreviations & Symbols

ACP Acyl carrier protein

Ala Alanine or alanylALA 웃-Aminolevulinic acid or웃-

aminolevulinate

amu Atomic mass unit (1.66 ⫻ 10⫺ 27kg or

1.66 ⫻ 10⫺ 24g)Arg Arginyl or arginylAsn Asparagine or asparaginylAsp Aspartic acid, aspartate, or aspartylAsx Aspartate ⫹ asparagine or aspartyl ⫹

asparaginylATCase Aspartate transcarbamoylaseATP Adenosine 5⬘-triphosphate

B Aspartate ⫹ asparagine (or

aspartyl ⫹ asparaginyl)BES N , N-Bis(2-hydroxyethyl)-2-

aminoethanesulfonic acid

Bi Two-substrate enzyme system

BPG D-2,3-BisphosphoglycerateBPTI Bovine pancreatic trypsin inhibitor

Cysteine or cysteinyl

CAP Catabolite gene activating proteincAPK Protein kinase A (or cyclic AMP-

stimulated protein kinase)CAPS 3-(Cyclohexylamino)propanesulfonic

acid

Abbreviations & Symbols

DPN⫹ see recommended abbreviation NAD⫹

DSC Differential scanning calorimetry

E Glutamic acid, glutamate, or glutamyl

EDTA Ethylenediaminetetraacetic acid or its

adenine dinucleotideFADH2 Reduced flavin adenine dinucleotideFBP Fructose 1,6-bisphosphate

Abbreviations & Symbols

GSH Glutathione (sometimes referred to as

reduced glutathione)GSSG Glutathione disulfide (sometimes

referred to as oxidized glutathione)GTP Guanosine 5⬘-triphosphate

H Histidine or histidyl

HMG-CoA 웁-Hydroxymethylglutaryl-CoA

Ile Isoleucine or isoleucyl

MES 2-(N-Morpholino)ethanesulfonic acid

MetHb Methemoglobin (or, Fe(III)Hb)MetMb Metmyoglobin (or, Fe(III)Mb)MOPS 3-(N-Morpholino)propanesulfonic acid

NANA N-Acetylneuraminic acid

Abbreviations & Symbols

NMR Nuclear magnetic resonance

NOESY Nuclear Overhauser effect

Phe Phenylalanine or phenylalanyl

PIP2 Phosphatidylinositol 4,5-bisphosphate

PIPES Piperazine-N,N⬘-bis(2-ethanesulfonic

acid)

PKA Protein kinase A (or cyclic

AMP-stimulated protein kinase)

Ubiqinone (Coenzyme Q or CoQ)

QCO2 The amount (in microliters) of CO2

given off (under standard conditions

of pressure and temperature) permilligram of tissue per hourQELS Quasi-elastic laser light scattering

propanesulfonic acid

Ter Three-substrate enzyme systemTES N-Tris(hydroxymethyl)methyl-2-

aminoethanesulfonic acid

Thr Threonine or threonylTIM or TPI Triose-phosphate isomeraseTPP Thiamin pyrophosphate (or thiamin

diphosphate)Tris Tris(hydroxymethyl)aminomethane

Transition state

TX Transition state intermediate

Abbreviations & Symbols

Tyr Tyrosine or tyrosyl

Uni One-substrate enzyme system

Val Valine or valyl

YADH Yeast alcohol dehydrogenase

Z Glutamate ⫹ glutamine or glutamyl ⫹

glutaminyl

Abbreviated Binding

Schemes

The following diagrams indicate the binding

interac-tions for enzyme kinetic mechanisms To conserve

space, the notation used in this Handbook is a compact

version of the diagrams first introduced by Cleland1 His

diagram for the Ordered Uni Bi Mechanism is as follows:

Throughout this handbook, we have used the following

single-line, compact notation:

EA앗 (EA}EPQ) 앗 P (EQ) 앗 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

un-bound (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

al-ways is indicated by a downward arrow, because this

systematic usage emphasizes the symmetry of certain

mechanisms The symbol ‘‘A앗’’ indicates that substrate

A adds; the symbol ‘‘A or B앗앗’’, ‘‘A or B or C앗앗앗’’,

etc , indicates random addition of two or three substrates,

respectively Interconversions of enzyme-bound

re-actants can be reversible (}) or irreversible (씮)

Taking the Ordered Uni Bi mechanism as an example,

we can consider several additional possibilities:

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

EA앗 (EA}EX}EPQ) 앗P (EQ) 앗Q

E

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

EA앗 (EA}EX 씮EPQ) 앗P (EQ) 앗Q

E

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

EA앗 (EA 씮EX 씮EPQ) 앗P (EQ)앗Q

where F}E represents the reversible isomerization step

Other examples of one-substrate and two-substrate netic mechanisms include:

ki-Ordered Uni Bi Mechanism

EA앗 (EA}EPQ) 앗P (EQ) 앗Q

Ordered Bi Bi Theorell-Chance Mechanism

EA앗 (EA) B앗앗P (EQ) 앗Q

E

Abbreviated Binding Schemes

Ping Pong Bi Bi Mechanism

EA앗 (EA}FP) 앗P (F) B앗 (FB}EQ) 앗Q

Source Words

A

A, B, C, /P, Q, R,

Symbols for substrates and products, respectively, in

multisubstrate enzyme-catalyzed reactions In all

or-dered reaction mechanisms, A represents the first

sub-strate to bind, B is the second, etc., whereas P denotes

the first product to be released, Q represents the second,

etc See Cleland Nomenclature

AB INITIO MOLECULAR-ORBITAL

CALCULATIONS

A method of molecular-orbital calculations for

de-termining bonding characteristics and other structural

information about a wide variety of compounds and

mo-lecular configurations, including those that may not be

directly observable (e.g., transition state configurations

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-cata-lyzed reactions Related methods, known as

semiempiri-cal methods, use simplifying assumptions in the semiempiri-

calcula-tions and are determined more quickly than standard ab

initiomethods

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.

ABM-1 & ABM-2 SEQUENCES IN

ACTIN-BASED MOTORS

Consensus docking sites1 for actin-based motility,

de-fined by the oligoproline modules in Listeria

monocyto-genesActA surface protein and human platelet

vasodila-tor-stimulated phosphoprotein (VASP) Analysis of

known actin regulatory proteins led to the identification

of distinct Actin-Based Motility (or Actin-Based-Motor)homology sequences:

ABM-1: (D/E)FPPPPX(D/E) [where X ⫽ P or T]ABM-2: XPPPPP [where X ⫽ G, A, L, P, or S]

Actin-based motility involves a cascade of binding actions designed to assemble actin regulatory proteins

inter-into functional locomotory units Listeria ActA surface

protein contains a series of nearly identical EFPPPPTDE-type oligoproline sequences for binding vasodila-tor-stimulated phosphoprotein (VASP) The latter, a tet-rameric protein with 20-24 GPPPPP docking sites, bindsnumerous molecules of profilin, a 15 kDa regulatoryprotein known to promote actin filament assembly2

Laine et al.3 recently demonstrated that proteolysis ofthe focal contact component vinculin unmasks an

ActA homologue for actin-based Shigella motility The

ABM-1 sequence (PDFPPPPPDL) is located at or nearthe C-terminus of the p90 proteolytic fragment of vin-culin Unmasking of this site serves as a molecular switchthat initiates assembly of an actin-based motility complexcontaining VASP and profilin Another focal adhesionprotein zyxin4 contains several ABM-1 homology se-quences that are also functionally active in reorganizingthe actin cytoskeletal network

1D L Purich & F S Southwick (l997) Biochem Biophys Res.

Comm 231, 686.

2F 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

as a synonym for ‘‘nonproductive complex’’ Complexes

that fail to undergo further reactions along the catalytic

pathway are called dead-end complexes, and the

reac-tions producing them are called dead-end reacreac-tions

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

en-zyme’s 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

nonpro-ductive complexes involving the binding of substrate(s)

and/or product(s) to one or more enzyme forms Thus,

nonproductive complexes that culminate in substrate

in-hibition are abortive complexes For a discussion

con-cerning formation of EB and EP complexes in

rapid-equilibrium ordered Bi Bi reactions, see the section on

the Frieden Dilemma Enzyme-substrate-product

com-plexes that often form with multisubstrate enzymes are

also abortive complexes

Early product inhibition studies of Aerobacter aerogenes

ribitol dehydrogenase5demonstrated the formation of the

E-NAD⫹-D-ribulose and E-NADH-D-ribitol complexes

In the lactate dehydrogenase reaction, the

E-NADH-lac-tate and E-NAD⫹-pyruvate complexes are stable6, and

determination of the Kd values indicates that the

E-NAD⫹-pyruvate ternary complex is physiologically

rele-vant7 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

bo-vine heart malate dehydrogenase7 Creatine kinase forms

an E-MgADP-creatine complex8 Inhibition at high

sub-strate-product concentrations may arise from factors

other than abortive complexes; for example, the

inhibi-tion observed in an equilibrium exchange experiment may

be related to high ionic strength of reaction solutions9

Wong and Hanes10 pointed out that equilibrium

ex-change studies can be useful in detecting the presence

of abortive species Although abortive complexes can

complicate exchange kinetic behavior, the Wedler-Boyer

protocol11minimizes the influence of abortives on

equi-librium exchange studies

Different abortives may be formed with alternative ucts or substrates Such procedures can be useful in help-ing to distinguish Theorell-Chance mechanisms from or-dered systems with abortive complexes12 In the case

prod-of lactate dehydrogenase, the E-pyruvate-NAD⫹and lactate-NADH abortive complexes may play a regula-

E-tory roles in aerobic versus anaerobic metabolism.

Computer simulations13also point to the regulatory tential of these non-productive complexes.See Deadend Complexes; Inhibition; Nonproductive Complexes; Product Inhibition; Substrate Inhibition; Isotope Trap- ping; Isotope Exchange at Equilibrium; Enzyme Regu- lation

po-1International Union of Biochemistry (1982) Eur J Biochem 28,

281.

2M Dixon & E C Webb (1979) Enzymes, 3rd ed., Academic Press,

New York.

3J D Allen (1979) Meth Enzymol 64, 248.

4H J Fromm (1975) Initial Rate Enzyme Kinetics, Springer-Verlag,

New York.

5H J Fromm & D R Nelson (1962) J Biol Chem 231, 215.

6H 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.

8E Silverstein & G Sulebele (1969) Biochemistry 8, 2543.

9J F Morrison & W W Cleland (1966) J Biol Chem 241, 673.

10J T.-F Wong & C S Hanes (1964) Nature 203, 492.

11F C Wedler & P D Boyer (1972) J Biol Chem 247, 984.

12C C Wratten & W W Cleland (1965) Biochemistry 4, 2442.

13D 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; tope exchange, 64, 32, 33, 39-45; isotope trapping, 64, 58; limita- tion, 63, 432-436; multiple, one-substrate system, 63, 473, 474;

iso-pH effects, 63, 205; practical aspects, 63, 477-480; substrate bition, 63, 500, 501; two-substrate system, 63, 474-478; in prod- uct inhibition studies, 249, 188-189, 193, 199-200, 205; identifica- tion of, 249, 188-189, 202, 206, 208-209.

inhi-ABSCISSA

The x-coordinate axis for a graph of Cartesian nates [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 Concentra- tion]-axis in so-called double-reciprocal or Lineweaver-Burk plots

coordi-ABSOLUTE CONFIGURATION

A method for designating the stereoisomeric tion of a chiral carbon atom within a molecular entity.The designationDwas arbitrarily assigned to (⫹)-glycer-aldehyde, and (⫺)-glyceraldehyde was assigned the label

configura-Absorption Coefficient

L Compounds that can be derived fromL-glyceraldehyde

without inversion reactions of the chiral center are

like-wise designatedL- (and, their mirror images,D-) X-ray

crystallographic studies later showed thatD

-glyceralde-hyde had the configuration shown below1.D- andL

-Ala-nine are also depicted

TheDL-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 Configuration;

Cahn-Ingold-Prelog System; Corn Rule; Diastereomers;

En-antiomers; (R/S)-Convention

1J M Bijuoet, A F Peerdeman & A J van Bommel (1951) Nature

168, 271.

2W 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 Configurations, Georg Thieme Publ., Stuttgart.

ABSOLUTE TEMPERATURE

A 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

super-script o used to indicate degree as on the Celsius scale

K has the same magnitude as degree Celsius (⬚C)

ABSOLUTE UNCERTAINTY

The 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

Uncer-tainty

ABSOLUTE ZERO

The 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

ABSORBANCE

A quantitative measure of photon absorption by a cule, expressed as the log10 of the ratio of the radiant

mole-intensity Ioof light transmitted through a reference

sam-ple 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 Chemistryrecommends that the definition should now be based on

the ratio of the radiant power of incident radiation (Po)

to the radiant power of transmitted radiation (P) Thus,

A ⫽ log(Po/P) ⫽ log T⫺ 1 In solution, Po would refer

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 definition assumes that light reflection

and light scattering are negligible If not, the appropriate

term for log(Po/P) is ‘‘attenuance.’’ See Beer-Lambert Law; Absorption Coefficient; Absorption Spectroscopy

ABSORBED DOSE

1 The quantity of absorbed energy absorbed per unitmass 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

phe-cess of transport of a substance into a cell

ABSORPTION COEFFICIENT

The log-base10attenuance (or absorbance) (i.e., D or A) divided by the optical pathlength (l ) This coefficient, symbolized by a, is thus equal to l⫺ 1log(Po/P) See Beer- Lambert Law; Absorbance; Molar Absorption Coeffi- cient; Absorption Spectroscopy

Absorption Spectroscopy

ABSORPTION SPECTROSCOPY

Absorption spectroscopy is widely used to follow the

course of enzyme-catalyzed reactions Absorbance

mea-surements should be made under conditions that permit

use of the Beer-Lambert Law:

A ⫽ log (Io/I) ⫽ ␧cl where A is the absorbance (a dimensionless parameter),

I0and I are the incident radiant intensity and the

trans-mitted radiant intensity, ␧ is the molar (base10)

absorp-tion coefficient, c is the molar concentraabsorp-tion of the

ab-sorbing species, and l is the absorption pathlength (i.e.,

the distance through solution that light must pass) [Note:

IUPAC1now favors A ⫽ log(Po/P) where Poand P are

the incident radiant power and the transmitted radiant

power, respectively.]

Figure 1 Perpendicular disposition of the electric vector E and

mag-netic vector H of a light wave traveling from its source in the direction

of propagation shown by the arrow Note that electromagnetic

radia-tion interacts with molecules in two ways: (a) in absorpradia-tion, 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.

Practical Considerations Typical absorption assay

meth-ods 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

cplots 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 fine cotton strands from tissue used to clean the faces

of cuvettes One is well advised to confirm the linearity of

absorbance with respect to product (or substrate)

concen-tration under the exact assay conditions to be employed in

rate experiments Prior centrifugation or filtration may beneeded to reduce light scattering or turbidity

Figure 2 Design features of a double-beam UV/visible tometer 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 compart- ment The chopper-motor rotates a beam-splitter that allows half of the in-coming light to travel to the reference compartment, while reflecting the other half of the in-coming light to the sample com- partment.

spectropho-Instruments with double monochromator configurations,

or equivalent multi-pass configurations, can greatly duce stray light (which is any radiation of wavelengthother than that of the columnated light beam) Ab-sorbance in the presence of stray light can be expressed as:

re-A ⫽ log [(Io⫹ Is)/(I ⫹ Is)]

where Isis the stray light intensity The larger the value

of Is(relative to Ioor I), the greater the error in

concen-tration or rate measurements2

Figure 3 Example of the Beer–Lambert relationship.

The Beer-Lambert relationship is additive (i.e., the

ab-sorption of light by one chemical species is unaffected

Absorption Spectroscopy

by the presence of other species, irrespective of whether

those other species absorb light at the same wavelength)

Thus, A ⫽ ⌺(␧icil) The greater the difference in the

molar absorption coefficients between the substrate(s)

and the product(s), the larger the change in absorbance

with time and the greater the ease in velocity

determina-tion This statement assumes that the two chemical

spe-cies do not interact with each other

Note that the Beer-Lambert relationship does not

re-quire one to monitor a reaction at the wavelength

maxi-mum 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 ␭maxvalue 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 ␧

Like-wise, one can use a wavelength other than 340 nm to

assay NADH or NADPH, but one should avoid

unneces-sary loss of signal-to-noise by working as close to the

wavelength yielding maximal absorption

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

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 prod- ucts that can be coupled to NAD ⫹ reduction or NADH oxidation.Occasionally, one can increase the ⌬␧ by utilizing alter-native substrates For example, 3-acetyl-NAD⫹or thio-NAD⫹can often be used with NAD⫹-dependent dehy-drogenases Note however that an alternative substratemay change the kinetic mechanism, as compared to thatobserved with the naturally occurring substrate Alterna-tive substrates are of particular value when the normalsubstrate(s) and product(s) do not efficiently absorb UV

or visible light For example, many nitroaniline or

p-nitrophenyl derivatives have proved to be quite useful

in enzyme assays because they exhibit intense absorptionaround 410 nm

Ideally, other components in the reaction mixture shouldnot absorb significantly at the monitored wavelength

In addition, colored impurities should be removed Forexample, commercial imidazole, a commonly usedbuffer, contains a yellow impurity that can be easilyremoved upon recrystallization from ethyl acetate

General Principles Light absorption is quantized The

energy change associated with an electronic transitionoccurs within the ultraviolet (UV) or visible (vis) spec-trum Visible light absorption corresponds to low-energyelectronic transitions, such as those observed with certaintransition metal ions or with molecules having conju-gated double bonds The near ultraviolet (200 and 400nm) corresponds to electronic transitions in molecular

entities with smaller conjugated systems (e.g., triene, ATP, etc.) Isolated double bonds, such as those

1,3,5-hexa-within peptide bonds, absorb light around 200-210 nm.Dioxygen, carbon dioxide, and water can absorb light of

Absorption Spectroscopy

wavelength less than 180 nm, and spectroscopy in the

far-UV 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 fine structure that become superimposed

on the potential energy surfaces of the electronic

transi-tions 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

propor-tional 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⫺ 1M⫺ 1) suggest that the transition is

accompa-nied 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

asso-ciated with UV-visible absorption spectroscopy consist

of an electron in a filled molecular orbital being excited

to the next higher energy orbital (an antibonding

or-bital) Although many exceptions are known, the relative

transition energies roughly are:␴ 씮 ␴* ⬎ n 씮 ␴* ⬎

n 씮 앟* ⬎ 앟 씮 앟*

␴ 씮 ␴* Transitions These transitions typically occur

between 120 and 220 nm (i.e., in the far-UV) The␭max

value for typical ␴ 씮 ␴* transitions of carbon-carbon

or carbon-hydrogen bonds is usually around 150 nm For

example, the␭maxvalue for ethane is 135 nm

n 씮 ␴* Transitions These transitions typically occur

at wavelengths greater than that needed for ␴ 씮 ␴*

transitions Roughly, n 씮␴* transitions involving UO¨ U

occur with wavelengths at about 185 nm, with UN¨U

and US¨U at about 195 nm, and with carbonyls at about

190 nm Examples of ␭max values (and ␧ values) of

n 씮␴* transitions include: water (␭max⫽ 167 nm with

␧ ⫽ 7000), methanol (␭max ⫽ 183 nm with ␧ ⫽ 500),

acetone (␭max ⫽ 188 nm with ␧ ⫽ 1860), and methyl

iodide (␭max⫽ 259 nm with ␧ ⫽ 400)

n 씮␲* Transitions These are ‘‘forbidden’’ transitions

according to symmetry rules, but molecular vibrations

allow these transitions to occur, albeit with low ties Nonbonding electrons of carbonyl groups will oftenhave n 씮 앟* transition ␭maxvalues of around 300 nm.Some examples include acetone (␭max ⫽ 279 nm, ␧ ⫽15), acetophenone (319 nm, ␧ ⫽ 50), thiourea (a shoulder

intensi-at 291 nm, ␧ ⫽ 71), and acetic acid (204 nm, ␧ ⫽ 41).The n 씮 앟* transition can also be detected in opticalrotatory dispersion measurements Moreover, n 씮 앟*transitions often exhibit a blue shift in polar solvents orenvironments

␲씮␲* Transitions Typically occurring at wavelengths

in the near-UV, transitions of this type are the mostcommonly utilized spectral signals in kinetic and struc-tural studies

1IUPAC (1988) Pure and Appl Chem 60, 1055.

2R D Allison & D L Purich (1979) Meth Enzymol 63, 3.

3C R Cantor & P R Schimmel (1980) Biophysical Chemistry, part

II, pp 344-408, Freeman, San Francisco.

4R P Bauman (1962) Absorption Spectroscopy, Wiley, New York.

5H H Jaffe & M Orchin (1962) Theory and Application of

Ultravio-let Spectroscopy, Wiley, New York.

Selected entries from Methods in Enzymology [vol, page(s)]:

Absorption Spectrophotometer: Application, 24, 15-25; line compensation, 24, 8-10; computerized, 24, 19-25; light scat- tering, 24, 13-15; monochromator, 24, 4; photometer, 24, 5-8; re- corder, 24, 8; sample compartment, 24, 5; single-beam, 24, 3-4; spectral characteristics, 24, 10-12; split-beam, 24, 3; stray light,

base-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; ning ultraviolet/visible spectroscopy applied in stopped-flow studies, 246, 168; transient absorption spectroscopy in the study

rapid-scan-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 tems, 226, 125; excitation source, 226, 121; global analysis, 226,

sys-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; he- moglobin data analysis, 232, 401-415; kinetic analyses, 232, 390; photoselection effects, 232, 390-391; kinetic intermediates and,

Acetate Kinase

232, 389; spectrometer for, 232, 392-401; performance

character-istics, 232, 389-390.

ABSORPTIVITY

A 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

refer-ence sample, and P is the radiant power being

transmit-ted through the solution The Commission on

Photo-chemistry does not recommend the use of this term

See Absorbance; Absorption Coefficient; Beer-Lambert

Law; Absorption Spectroscopy

ABSTRACTION REACTION

Any 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

stereo-chemistry

ACCELERATION

In physics, the time rate of change of motional velocity

resulting from changes in a body’s speed and/or

direc-tion 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;

Ca-talytic Proficiency; Efficiency Function

ACCRETION

Solute 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

ACCURACY

The closeness or proximity of a measured value to the

true value for a quantity being measured Unless the

magnitude of a quantity is specified by a formal SI

defini-tion, one typically uses reference standards to establish

the accepted true value for a given quantity See also Precision

ACETALDEHYDE DEHYDROGENASE (ACETYLATING)

This enzyme [EC 1.2.1.10] catalyzes the oxidation ofacetaldehyde in the presence of NAD⫹and coenzyme

A to form acetyl-CoA ⫹ NADH ⫹ H⫹ Other aldehydesubstrates include glycolaldehyde, propanal, and bu-tanal

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.

2-(ACETAMIDOMETHYLENE)SUCCINATE HYDROLASE

This enzyme [EC 3.5.1.29] catalyzes the hydrolysis of (acetamidomethylene)-succinate to yield acetate, succi-nate semialdehyde, carbon dioxide, and ammonia

2-R W Burg (1970) Meth Enzymol 18(A), 634.

is sequential and that there is direct in-line phosphoryltransfer Incidental generation of a metaphosphate anionduring catalysis may explain the formation of an enzyme-bound acyl-phosphate Acetate kinase is ideally suitedfor the regeneration of ATP or GTP from ADP orGDP, respectively

1P A Frey (1992) The Enzymes 20, 160.

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-

Acetate Kinase

ing, 63, 275-278; metaphosphate, 87, 12, 20; metaphosphate

syn-thesis and, 6, 262-263; nucleoside diphosphate kinase activity;

63, 8; phosphorylation potential, 55, 237; pH stability profile, 87,

18; promoting microtubule assembly, 85, 417-419; purine

nucleo-side diphosphate kinase activity, 63, 8; regenerating GTP from

GDP, 85, 417; ribulose-5-phosphate 4-epimerase and, 5,

253-254; Veillonella alcalescens acetate kinase [ATP formation assay,

71, 312; hydroxamate assay, 71, 311; properties, 71, 315;

stabil-ity to heat, 71, 313; stimulation by succinate, 71, 316; substrate

specificity, 71, 316]; xylulose-5-phosphate 3-epimerase and, 5,

250-251; xylulose-5-phosphate phosphoketolase and, 5, 26;

pu-rine inhibitor, 63, 398; metal-ion binding, 63, 275-278;

phospho-rothioates, 87, 200, 205, 226, 232, 258; from Bacillus

stearother-mophilus; assay, 90, 179; properties, 90, 183; purification, 90,

180; in acetyl phosphate and acetyl-CoA determination, 122, 44;

and hexokinase, in glucose 6-phosphate production, 136, 52;

di-hydroxyacetone phosphate synthesis with, 136, 277; glucose

6-phosphate synthesis with, 136, 279; sn-glycerol 3-6-phosphate

syn-thesis with, 136, 276; in pyruvic acid phosphoroclastic system,

243, 96, 99.

ACETATE KINASE (PYROPHOSPHATE)

This enzyme [EC 2.7.2.12] converts acetate and

pyro-phosphate to form acetyl pyro-phosphate and

orthophos-phate

H G Wood, W E O’Brien & G Michaels (1977) Adv Enzymol 45,

8555.

ACETAZOLAMIDE

A diuretic agent

(5-acetamido-1,3,4-thiadiazole-2-sul-fonamide) that acts as a potent noncompetitive inhibitor

(Ki10⫺ 8M) of carbonic anhydrase

ACETOACETATE DECARBOXYLASE

This enzyme [EC 4.1.1.4] catalyzes the decarboxylation

of acetoacetate to form acetone and carbon dioxide

M H O’Leary (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 SYNTHASE

This enzyme [EC 4.1.3.18] catalyzes the reversible

car-boxylation of 2-acetolactate with carbon dioxide to form

two pyruvate ions Thiamin pyrophosphate is a

re-quired cofactor

B A Palfey & V Massey (1998) Comprehensive Biological

Cataly-sis: A Mechanistic Reference 3, 83.

R L Schowen (1998) Comprehensive Biological Catalysis: A

Mecha-nistic Reference 2, 217.

J V Schloss & M S Hixon (1998) Comprehensive Biological

Cataly-sis: A Mechanistic Reference 2, 43.

A Schellenberger, G Hu¨bner & H Neef (1997) Meth Enzymol.

279, 131.

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.

ACETYLCHOLINESTERASE

This enzyme [EC 3.1.1.7], also known as true ase, choline esterase I, and cholinesterase, catalyzes thehydrolysis of acetylcholine to produce choline and ace-tate The enzyme will also act on a number of acetateesters as well as catalyze some transacetylations

cholinester-D M Quinn & S R Feaster (1998) Comprehensive Biological

Catal-ysis: 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; modification by symmetrical disul- fide radical, 251, 100; thioester substrate, 248, 16; transition state and multisubstrate analogues, 249, 305; enzyme receptor, similarity to collagen, 245, 3.

ACETYL-CoA (or, ACETYL COENZYME A)

As the principal thiolester of intermediary metabolism,acetyl coenzyme A is involved in two-carbon biosyn-thetic and degradative steps An essential component isthe vitamin pantithenic acid, which provides the sulfuratom 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; traction 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

ex-synthetase, 13, 375; N-acetyltransferase, 17B, 805; aminoacetone

N-Acetylgalactosaminide Sialyltransferase

synthase, 17B, 585; carnitine acetyltransferase, 13, 387-389; 14,

613; choline acetyltransferase, 17B, 780, 788, 798; citrate

syn-thase, 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 C-ACETYLTRANSFERASE

(or, THIOLASE)

This enzyme [EC 2.3.1.9], also known as thiolase,

trans-fers 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-ACYLTRANSFERASE

This enzyme [EC 2.3.1.16], also known as

3-ketoacyl-CoA thiolase, transfers an acyl group from an acyl-3-ketoacyl-CoA

to acetyl-CoA to form free coenzyme A and

3-oxoa-cyl-CoA

J V Schloss & M S Hixon (1998) Comprehensive Biological

Cataly-sis: A Mechanistic Reference 2, 43.

ACETYL-CoA:ACP TRANSACYLASE

This enzyme [EC 2.3.1.38], also referred to as

acetyl-CoA:[acyl-carrier protein] S-acetyltransferase, transfers

an acetyl group from one acetyl-CoA to an

acyl-carrier-protein (ACP) to form free coenzyme A and the

acetyl-[acyl-carrier-protein].See also Fatty Acid Synthase

S 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.

ACETYL-CoA CARBOXYLASE

This enzyme [EC 6.4.1.2] catalyzes the reaction of

acetyl-CoA, bicarbonate, and ATP to form malonyl-acetyl-CoA,

or-thophosphate, 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

Cataly-sis: 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.

L A Kleczkowski (1994) Ann Rev Plant Physiol Plant Mol Biol.

This enzyme [EC 4.2.1.71] adds water to propynoate toform 3-hydroxypropenoate The enzyme will also act on3-butynoate to form acetoacetate

J V Schloss & M S Hixon (1998) Comprehensive Biological

Cataly-sis: A Mechanistic Reference 2, 43.

N-ACETYLGALACTOSAMINE-4-SULFATE

SULFATASE

This enzyme [EC 3.1.6.12] acts on 4-sulfate groups of the

N-acetylgalactosamine 4-sulfate moieties in chondroitinsulfate and dermatan sulfate

H Kresse & J Glo¨ssl (1987) Adv Enzymol 60, 217.

N-ACETYLGALACTOSAMINE-6-SULFATE

SULFATASE

This enzyme [EC 3.1.6.4] acts on 6-sulfate groups of the

N-acetylgalactosamine 6-sulfate moieties in chondroitinsulfate and the galactose 6-sulfate groups in keratansulfate

H Kresse & J Glo¨ssl (1987) Adv Enzymol 60, 217.

N-ACETYLGALACTOSAMINIDE

SIALYLTRANSFERASE

This 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-움-N-acetylneuraminyl)-(N-acetylgalactos-

aminyl)-glycoprotein

T A Beyer, J E Sadler, J I Rearick, J C Paulson & R L Hill

(1981) Adv Enzymol 52, 23.

N-Acetylglucosamine Kinase

N-ACETYLGLUCOSAMINE KINASE

This 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.

N-ACETYLGLUCOSAMINE-6-PHOSPHATE

2-EPIMERASE

This enzyme catalyzes the epimerization at the 2-position

of N-acetylglucosamine 6-phosphate See also

N-Acyl-glucosamine-6-phosphate 2-Epimerase

M E Tanner & G L Kenyon (1998) Comprehensive Biological

Ca-talysis: A Mechanistic Reference 2, 7.

N-ACETYLGLUCOSAMINE-6-SULFATE

SULFATASE

This 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 Glo¨ssl (1987) Adv Enzymol 60, 217.

␣ -N-ACETYLGLUCOSAMINIDASE

This enzyme [EC 3.2.1.50] catalyzes hydrolysis of

termi-nal nonreducing N-acetylglucosamine residues in

N-ace-tyl-움-glucosaminides

H Kresse & J Glo¨ssl (1987) Adv Enzymol 60, 217.

␤ -N-ACETYLGLUCOSAMINIDASE

This enzyme, reportedly catalyzing the hydrolysis of

ter-minal, nonreducing N-acetyl-웁-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-ACETYLGLUTAMATE SYNTHASE

This enzyme [EC 2.3.1.1], also referred to as

amino-acid acetyltransferase and acetyl-CoA : glutamate

N-acetyltransferase, catalyzes the reaction of acetyl-CoA

with glutamate to form coenzyme A and

N-acetylgluta-mate The enzyme will also acts on aspartate and, more

slowly, with some other amino acids The mammalian

enzyme is activated byL-arginine.See also Glutamate

Acetyltransferase

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 REDUCTASE

This enzyme [EC 1.2.1.38], also known as

N-acetylgluta-mate semialdehyde dehydrogenase and NAGSA

dehy-drogenase, 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-ACETYLHEXOSAMINIDASE

This enzyme [EC 3.2.1.52], also referred to as

웁-hexos-aminidase and N-acetyl-웁-glucos웁-hexos-aminidase, catalyzes the hydrolysis of terminal nonreducing N-acetylhexosamine residues in N-acetyl-웁-hexosaminides N-Acetylgluco- sides and N-acetylgalactosides are substrates.

H Kresse & J Glo¨ssl (1987) Adv Enzymol 60, 217.

H M Flowers & N Sharon (1979) Adv Enzymol 48, 29.

methio-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-ACETYLNEURAMINATE LYASE

This enzyme [EC 4.1.3.3], also known as raminate aldolase, will convert N-acetylneuraminate to

N-acetylneu-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

Mechanis-tic Reference 2, 135.

W A Wood (1972) The Enzymes, 3rd ed., 7, 281.

N2-ACETYLORNITHINE AMINOTRANSFERASE

This enzyme [EC 2.6.1.11] catalyzes the phate-dependent reaction of 2-acetylornithine with 움-

pyridoxal-phos-Acid Catalysis

ketoglutarate to produce N-acetylglutamate

5-semialde-hyde 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.

N2-ACETYLORNITHINE DEACETYLASE

This enzyme [EC 3.5.1.16], also known as

acetylornithi-nase and N-acetylornithiacetylornithi-nase, catalyzes the reaction of

water with N2-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)-LYASE

This enzyme [EC 4.2.99.8], also known as cysteine

syn-thase and O-acetylserine sulfhydrylase, catalyzes the

pyr-idoxal-phosphate-dependent reaction of H2S with O3

-acetylserine 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.

ACHIRAL

The absence of chirality Achiral molecules have an

in-ternal plane or point of symmetry A molecular

configu-ration is said to be achiral when it is superimposable on

its mirror image.See Chirality

ACID

A substance that liberates protons as a consequence of its

dissolution or dissociation.See Brønsted Theory; Lewis

Acid; Lewis Base

ACID-BASE EQUILIBRIUM CONSTANTS

The following table contains pK values for a selected

group of biochemically important substances The

asso-ciated thermodynamic parameters and the original

liter-ature references were presented by Edsall and Wyman1

pKaValues for Selected Substances at 25ⴗC

1J T Edsall & J Wyman (1958) Biophysical Chemistry, pp 452,

Aca-demic Press, New York.

ACID CATALYSIS

Any process for which the rate of reaction is acceleratedthrough the participation of an acid as a catalyst See General Acid Catalysis; Specific Acid Catalysis

ACIDITY

1 The tendency for a Brønsted acid to act as a proton

donor, expressed in terms of the compound’s 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 Brønsted 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;

Acid-Base Equilibrium Constants; Brønsted Theory; Lewis

Acid; Acidity Function; Leveling Effect

ACIDITY FUNCTION

A 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)1–3

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 specified), Hois defined to be

pKBHW ⫹⫺ log([BH⫹]/[B]) The value of Hois measured

using a weak indicator base whose pK value in water is

known Two common indicators are the aniline

deriva-tives, 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

mea-sured, usually spectrophotometrically, in the given

sol-vent Knowledge of a substance’s pK in water (i.e.,

pKBHW ⫹) allows the investigator to calculate Ho Once

Hois known, pKavalues can be determined for any other

acid-base pair

The value of hois defined as aH ⫹fI/fHI ⫹where aH ⫹is the

chemical activity of the proton, and fIand fHI ⫹are the

activity coefficients of the indicator Hoand hoare related

by Ho⫽ ⫺log ho For dilute solutions, Ho⫽ pH This

is the situation for most biochemical reactions The script is a reference to the net charge on the base Hence,

sub-H⫺is the corresponding acidity function for tively charged bases in equilibrium with neutral acids

mononega-The Hammett acidity function only applies to acidic vents having a high dielectric constant, further requiring

sol-that the fI/fHI ⫹ratio be independent of the nature of theindicator Thus, the Hammett acidity function is applica-ble for uncharged indicator bases that are aniline deriva-tives As mentioned above, other acidity functions havebeen suggested No single formulation has been devel-oped that satisfies different solvent systems or types ofbases

Bunnett and Olsen6–8used a different approach and rived the equation:

de-log([SH⫹]/[S]) ⫹ Ho⫽ ⌽(Ho⫹ log[H⫹]) ⫹ pKSH ⫹

in which S is a base that can be protonated by an acidicsolvent Plotting log([SH⫹]/[S]) ⫹ Ho versus Ho ⫹log[H⫹] will result in a reasonably linear line having aslope of ⌽ The method uses a linear free energy relation-ship to address the problems of defining basicity for weakorganic bases in solutions of moderate concentrations ofmineral acids The value and sign of ⌽ is a measure ofthe response of the acid-base equilibria to changes inacid concentration An analogous equation for kineticdata is

log k⌿⫹ Ho⫽ ⌽(Ho⫹ log[H⫹]) ⫹ log k2o

where k␺ is the pseudo-first-order rate constant for a

reaction in acidic solution and k2ois the correspondingconstant at infinite dilution in water Here ⌽ is suggested

to represent the response of the chemical reactionrate to changes in the acid concentration Attempts havebeen made to apply this method to basic media More-O’Ferrell9suggested that the slope may be related semi-quantitatively to the structure of the transition state

A related treatment of the acidity function considerschanges in acidity of the solvent on an acid-base equilib-ria10 With this procedure, an equilibrium is chosen as

reference, having an equilibrium constant K⬘o for the

reference reaction in a reference solvent and K⬘ for the

reference reaction in the particular medium under study

The reaction under study has the corresponding

equilib-ria of Ko(in the reference solvent) and K (in the

particu-lar medium) Thus,

log (K⬘/Ko⬘) ⫽ m* log (K/Ko)

where the slope m* corresponds to 1 ⫺ ⌽ in the

Bunnett-Olsen treatment Bunnett11has also plotted log k␺⫹ Ho

vs log awaterwhere awateris the activity of water and has

indicated that the slope of the line, w, suggests certain

possibilities for the chemical reaction mechanism in

mod-erately 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 Bakule12have criticized

this approach.See also Degree of Dissociation;

Hender-son-Hasselbalch Equation; Acid-Base Equilibrium

Con-stants; Bunnet-Olsen Equations; Cox-Yeats Treatment

1C H Rochester (1970) Acidity Functions, Academic Press, New

York.

2J March (1985) Advanced Organic Chemistry, Wiley, New York.

3J Hine (1962) Physical Organic Chemistry, McGraw-Hill, New York.

4L Zucker & L P Hammett (1939) J Amer Chem Soc 61, 2791.

5L P Hammett (1970) Physical Organic Chemistry McGraw-Hill,

8 V Lucchini, G Modena, G Scorrano, R A Cox & Y Yates (1982)

J Am Chem Soc 104, 1958.

9R A More O’Ferrall (1972) J Chem Soc., Perkin Trans 2, 976.

10A Bagno, G Scorrano & R A More O’Ferrall (1987) Rev Chem.

Interm 7, 313.

11J F Bunnett (1961) J Amer Chem Soc 83, 4956.

12F A Long & R Bakule (1963) J Amer Chem Soc 85, 2313.

ACID-LABILE SULFIDES

The bridging sulfur atoms in iron-sulfur proteins are

often referred to as acid-labile sulfides, because

treat-ment of such proteins with acids generates H2S

ACID PHOSPHATASE

This enzyme [EC 3.1.3.2], also referred to as acid

phos-phomonoesterase, phosphos-phomonoesterase, and

glycero-phosphatase, catalyzes the hydrolysis of an

orthophos-phoric monoester to generate an alcohol and

orthophosphate The enzyme, which has a wide ity, will also catalyze transphosphorylations

specific-M Cohn (1982) Ann Rev Biophys Bioeng 11, 23.

V P Hollander (1971) The Enzymes, 3rd ed., 4, 449.

ACONITASE

This [4Fe-4S] cluster-containing enzyme [EC 4.2.1.3],also known as citrate hydro-lyase and aconitate hydra-

tase, 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

J V Schloss & M S Hixon (1998) Comprehensive Biological

Cataly-sis: A Mechanistic Reference 2, 43.

H Lauble, M C Kennedy, H Beinert & D C Stout

(1992)Biochem-istry 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.

ACONITATE DECARBOXYLASE

This enzyme [EC 4.1.1.6] catalyzes the conversion of

cis-aconitate to itaconate (or, 2-methylenesuccinate) andcarbon dioxide

J V Schloss & M S Hixon (1998) Comprehensive Biological

Cataly-sis:A Mechanistic Reference 2, 43.

R Bentley (1962) Meth Enzymol 5, 593.

ACONITATE ⌬-ISOMERASE

This enzyme [EC 5.3.3.7] catalyzes the interconversion

of trans-aconitate to cis-aconitate, the reaction

report-edly to occur by an allelic rearrangement

D J Creighton & N S R K Murthy (1990) The Enzymes, 3rd ed.,

19, 323.

ACROSIN

This enzyme [EC 3.4.21.10] catalyzes the hydrolysis ofArg-Xaa and Lys-Xaa peptide bonds The enzyme be-longs to the peptidase family S1 and is inhibited by natu-rally occurring trypsin inhibitors

J S Bond & P E Butler (1987) Ann Rev Biochem 56, 333.

Actin Assembly Assays

ACTIN ASSEMBLY ASSAYS

The 43 kDa actin monomer (often termed globular actin

or G-actin) polymerizes to form filamentous (or F-) actin,

a process that can be measured by a number of

biochemi-cal and biophysibiochemi-cal 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

advan-tages and disadvanadvan-tages of each of these techniques

Table I

Methods to Measure Actin Polymerization

Method ␣ [polymer] ratio to length Shear rate actin Expense size (ml)

4 Fluorescence

(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 orifice of

a glass capillary viscometer (2) Difference spectroscopy

at 232 nm can be utilized to estimate the amount of

F-actin 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 filaments with the direction of

flow imparted by the rotation of one of two concentric

cylinders containing the filaments 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 the actin

monomer (5) Light scattering can be used to study

poly-merization because the filaments scatter light much more

intensely than individual actin subunits The 90⬚ light

scattering intensity is directly related to polymer weight

concentration (6) Electron microscopy can provide a

direct assessment of the length of suitably fixed and

contrast-stained actin filaments (7) DNase inhibition of

actin polymerization takes advantage of the observationthat DNase I preferentially binds to actin monomerswith sufficient affinity to block any polymerization of

DNase-actin complex (8) Pelleting relies on the much

higher sedimentation coefficient of actin filaments as

compared to monomeric actin (9) Millipore filtration assays allow one to rapidly separate monomeric andpolymeric actin using filter disks with 0.45 애m pores

Also see Protein Polymerization; Self-Assembly anisms.

Mech-1J A Cooper & T D Pollard (1982) Meth Enzymol 85, 182.

2F Oosawa & S Asakura (1975) Thermodynamics of Protein

Poly-merization, Academic Press, New York.

ACTIN ASSEMBLY KINETICS

The self-assembly of actin filaments occurs through quential head-to-tail polymerization, a process that pas-ses through phases called nucleation, elongation, andestablishment of the monomer-polymer equilibrium,polymer length redistribution, and treadmilling1-3 Thelast two are properties of assembled filaments Mono-meric actin is called G-actin (or globular actin), andpolymeric actin is termed F-actin (or filamentous actin).The most extensively studied actins are those obtained in

se-high abundance from rabbit muscle and Acanthamoeba castellani Many features of actin polymerization areanalogous to tubulin, and the reader may wish to consultthat entry See Microtubule Assembly Kinetics.

Actin Assembly Kinetics

Figure 1 Nucleation and growth of actin filaments Nucleation is

shown here as a thermodynamically unfavored process, which in the

presence of sufficient actin-ATP will undergo initial elongation to

form small filament structures that subsequently elongate with rate

constants that do not depend on filament length Elongation

pro-ceeds until the monomeric actin (or G-actin) concentration equals

the critical concentration for actin assembly.

Figure 2 Scheme for the addition and loss of actin-ATP or actin-ADP from the barbed and pointed ends of an actin filament The barbed end

is the faster growing and more stable end of an actin filament While the exchange of actin-ADP with ATP to yield actin-ATP and ADP is shown here as a spontaneous process, the actin regulatory protein profilin greatly accelerates the exchange process Note also that hydrolysis is thought to occur after (and not coincident with) addition of actin-ATP at either end.

Nucleation is the most thermodynamically unfavorablestep (Fig 1) in which three actin⭈ATP molecules mustcome together to form a polymerization nucleus Onlywhen the actin⭈ATP concentration is sufficiently highcan these unstable nuclei persist at sufficient concentra-tions for polymerization to occur Uncoordinated spon-taneous nucleation must be suppressed in certain re-gions of the cytoplasm, and actin monomer-sequesteringproteins may limit nucleation by decreasing the avail-able concentration of actin⭈ATP monomers TheADP⭈actin complex appears to be a potent inhibitor

of nucleation The nucleation process cannot persistfor long if elongation is occurring Indeed, if Atotal isthe total monomer concentration at the beginning ofelongation, then the rate of spontaneous nucleationwill be proportional to the third power of Atotal:

RateNucleation ⫽ kN[Atotal]3

and a 20% drop in Atotalshould reduce the rate of ation by a factor of two:

nucle-Rate0.8/Rate1.0⫽ kN[0.8Atotal]3/kN[Atotal]3

⫽ (0.8)3[Atotal]3/[Atotal]3⫽ 0.5

This effect will be even greater if there is any ADP-actin

in the total pool of actin monomers, especially if actin inhibits nucleation

ADP-Actin Assembly Kinetics

Elongation is the repetitive addition reactions of

actin⭈ATP (Fig 2) The actin-bound ATP is hydrolyzed

during/after monomer addition to filaments, forming

polymer-bound ADP and releasing orthophosphate The

kinetics of elongation conform to that predicted by the

following rate law:

dCp/dt ⫽ k⫹N[Atotal] ⫺ k⫺ 1N

where Cp is the concentration of polymerized actin, k⫹

is the bimolecular rate constant (equal to the sum of the

on-rate constants for actin monomer addition to both

ends of an actin filament, N is the number concentration

of polymer ends that are capable of reacting with actin

monomers, [Atotal] is the total monomer concentration,

and k⫺ 1 is the rate constant for monomer dissociation

If N is constant during the elongation phase, the observed

rate process will fit a simple first-order decay curve

Elongation continues until the elongation ‘‘on’’-rate

(i.e., k⫹[actin]) is exactly balanced by the ‘‘off’’-rate (k⫺)

This condition defines the critical actin concentration

([actin]critical⫽ k⫺/k⫹), a parameter which represents the

concentration of actin that coexists with assembled

fila-ments (Fig 3, top)

Because the onset of monomer-polymer equilibrium can

occur before the filaments achieve their own equilibrium

concentration behavior, these filaments will undergo

polymer length redistribution This is a slow process in

vitrothat in many respects resembles crystallization (See

Ostwald Ripening).

Treadmilling is the net opposite-end

assembly/disassem-bly process first described by Wegner’s study4 of actin

polymer dynamics He recognized that there was no

rea-son to believe that the release of free energy of ATP

hydrolysis need be identical for addition reactions

oc-curring on opposite filament ends The so-called

(⫹)-end and (⫺)-(⫹)-end have different rate and equilibrium

As the name implies, actin treadmilling does not involve

any net increase in the amount of polymerized actin;

instead, when ATP is present, monomers are

spontane-Figure 3 Critical concentration behavior of actin self-assembly For the top diagram depicting the macroscopic critical concentration curve, one determines the total amount of polymerized actin by meth- ods that measure the sum of addition and release processes occurring

at both ends Examples of such methods are sedimentation, light scattering, fluorescence assays with pyrene-labeled actin, and viscos- ity measurements For the bottom curves, the polymerization behavior

is typically determined by fluorescence assays conducted under ditions where one of the ends is blocked by the presence of molecules such as gelsolin (a barbed-end capping protein) or spectrin-band 4.1- actin (a complex prepared from erythrocyte membranes, such that only barbed-end growth occurs) Note further that the barbed end (or (⫹)-end) has a lower critical concentration than the pointed end (or (⫺)-end) This differential stabilization requires the occurrence of ATP hydrolysis to supply the free energy that drives subunit addition

con-to the (⫹)-end at the expense of the subunit loss from the (⫺)-end.

ously released from the less stable (⫺)-ends, and mers are taken up by the more stable (⫹)-ends (Fig 3).This results in a flux of monomers that can be quantita-tively assessed by the inclusion of [3H]ATP or [14C]ATPwhich exchanges with unlabeled actin in the monomericpool and upon polymerization is trapped in filaments asactin-ADP

mono-Actin-Based Pathogen Motility

Although investigations of the in vitro polymerization

process have provided a theoretical underpinning for

probing intracellular actin dynamics, there are obvious

voids in our understanding Actin-based motility in many

cell types reaches a rate of 1 애m/second, corresponding

to the steady-state addition/loss of 50 to 100 monomers

per filament per second This is much faster than any in

vitrokinetic study, and the mechanism of assembly may

differ substantially from the model studies For example,

although exchange of ATP for ADP on actin is not a

rate-limiting reaction in vitro, this may not be true in

the cell Intracellular F-actin formation is also highly

localized, occurring within discrete polymerization zones

established by assembly of actin-based motor complexes

from a battery of regulatory proteins These include

cap-ping proteins, severing proteins, sequestering proteins,

bundling and cross-linking proteins, actin-related

pro-teins, as well as a growing list of actin-based motor

com-plex components Their interactions are controlled by

binding interactions (See Actin Filament Capping

Pro-tein, Actin Filament Severing ProPro-tein, Actin Filament

Bundling/Cross-Linking Protein, and ABM-1 & ABM-2

Sequences in Actin-Based Motors) In addition, actin

assembly is controlled by extracellular cues transmitted

to the cell’s interior by focal adhesion and adherens

junction proteins

1T P Stossel (1993) Science 260, 1086.

2M F Carlier (1989) Int Rev Cytol 115, 139.

3T D Pollard & J A Cooper (1986) Annual Rev Biochem 55, 987.

4A Wegner (1982) J Mol Biol 161, 607.

Selected entries from Methods in Enzymology [vol, page(s)]:

Staining with fluorochrome-conjugated phalloidin, 194, 729;

bind-ing activity of ponticulin, 196, 58; crosslinkbind-ing in

agonist-stimu-lated cells, assay, 196, 486; depolymerization [actin-binding

pro-tein effects, 215, 74; myosin effects, 215, 74; dilution effect on

platelet cytoskeleton, 215, 76]; expression in Escherichia coli,

196, 368 [growth conditions for, 196, 378; solubility, effect of

bacterial lysis, 196, 380; vector-related variability, 196, 370];

fluo-rescein labeling, 196, 50; actin-gelsolin complex [assay, 215, 94;

isolation from platelets, 215, 89]; globular, purification from

Dic-tyostelium discoideum, 196, 89; isoforms, separation, 196, 105;

monomeric columns with, preparation, 196, 310; nucleation

activ-ity [agonist-stimulated, assay of inhibitor, 196, 495;

measure-ment, 196, 493]; platelet [characterization, 215, 58; purification,

215, 58, 66; recombination with actin-binding protein and

움-ac-tinin, 215, 73]; polymerization in agonist-stimulated cells, assay,

196, 486; preparation, 196, 402; actin-profilin complex, isolation

from cell extracts, 196, 97; purification, 196, 50, 74; separation

from profilin:actin, 196, 115; skeletal muscle, labeling with

py-rene, 196, 138; sliding over myosin-coated surfaces, assay, 196,

399; stored, rejuvenation by recycling, 196, 403.

Actin filaments: affinity column [preparation, 196, 49; properties,

196, 305]; assay [in cells, 196, 486; in platelet lysates, 215, 54];

associated proteins, identification, 215, 50; attachment to

col-umn matrix, 196, 309; chromatography, 196, 312; crosslinking in cells, measurement, 196, 491; fluorescent labeling, 196, 403; im- aging, 196, 408; length measurement, 196, 416; proteins bind- ing, affinity chromatography, 196, 303.

Actin-binding proteins: ABP-50, purification, 196, 78; ABP-120, rification, 196, 79; ABP-240 purification, 196, 76; effect on actin depolymerization, 215, 74; extraction, 196, 311; isolation from

pu-Dictyostelium discoideum, 196, 70; platelet-derived actin

bind-ing proteins [characterization, 215, 58; purification, 215, 58, 64;

recombination with actin, 215, 73]; 30-kDa Dictyostelium

dis-coideum actin-crosslinking protein [assays, 196, 91; preparation,

196, 84]; actin-depolymerizing factor [assay, 196, 132]; DNase assay, 196, 136; platelet-derived a-actinin [characterization, 215, 58; purification, 215, 58, 70; recombination with actin, 215, 73].

ACTIN ATPase REACTION (Apparent Irreversibility)

The pathway of ATP hydrolysis associated with rabbitmuscle actin polymerization was investigated using anassay of intermediate 18O-positional isotope exchangereactions Under a variety of conditions that influencethe rate and extent of F-actin self-assembly, ATP hydro-lysis proceeded without any evidence of multiple rever-sals that are characteristic of any reversible phosphoan-hydride-bond cleavage1 These results are in harmonywith published findings2indicating that GTP hydrolysisduring tubulin polymerization also fails to display anyevidence of intermediate exchange reactions

1 M F Carlier, D Pantaloni, J A Evans, P K Lambooy, E D.

Korn & M R Webb (1988) FEBS Lett 235, 211.

2J M Angelastro & D L Purich (1990) Eur J Biochem 191, 507.

ACTIN-BASED PATHOGEN MOTILITY

Actin-based motility involves the highly regulated sembly/disassembly of actin filaments in the cytoplasmnearest the peripheral membrane The energetics andpolarity of ATP-dependent filament assembly producethe expansive force and directionality for peripheralmembrane protrusion during ameboid motion1 Twobacterial systems bypass the complexities of signal trans-

as-duction cascades as well as membrane geometry: Listeria monocytogenes, a gram-positive rod that causes severe

meningitis and bacteremia, and Shigella flexneri, a

gram-negative rod that is a leading cause of bacillary dysentery

Listeriaproduces proteins known as internalins to inducephagocytosis2,3, and Shigella uses the surface proteins,

Ipa B and C4 Although most bacteria are trapped and

killed in phagolysosomes, ingested Listeria and Shigella

readily escape by producing hemolysins that disrupt thephagolysosomal membrane5-7 After entering the host