Enzymatic Transition States, Transition-State Analogs ... · BI80CH29-Schramm ARI 20 May 2011 13:26...

BI80CH29-Schramm ARI 20 May 2011 13:26

Enzymatic Transition States,Transition-State Analogs,Dynamics, Thermodynamics,and LifetimesVern L. SchrammDepartment of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461;email: [email protected]

Annu. Rev. Biochem. 2011. 80:703–32

The Annual Review of Biochemistry is online atbiochem.annualreviews.org

This article’s doi:10.1146/annurev-biochem-061809-100742

Copyright c© 2011 by Annual Reviews.All rights reserved

0066-4154/11/0707-0703$20.00

Keywords

drug design, inhibitor design, isotope effects, protein conformations,protein dynamics

Abstract

Experimental analysis of enzymatic transition-state structures useskinetic isotope effects (KIEs) to report on bonding and geometrydifferences between reactants and the transition state. Computationalcorrelation of experimental values with chemical models permits three-dimensional geometric and electrostatic assignment of transition statesformed at enzymatic catalytic sites. The combination of experimentaland computational access to transition-state information permits(a) the design of transition-state analogs as powerful enzymaticinhibitors, (b) exploration of protein features linked to transition-statestructure, (c) analysis of ensemble atomic motions involved in achievingthe transition state, (d ) transition-state lifetimes, and (e) separationof ground-state (Michaelis complexes) from transition-state effects.Transition-state analogs with picomolar dissociation constants havebeen achieved for several enzymatic targets. Transition states of closelyrelated isozymes indicate that the protein’s dynamic architecture islinked to transition-state structure. Fast dynamic motions in catalyticsites are linked to transition-state generation. Enzymatic transitionstates have lifetimes of femtoseconds, the lifetime of bond vibrations.Binding isotope effects (BIEs) reveal relative reactant and transition-state analog binding distortion for comparison with actual transitionstates.

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Contents

INTRODUCTION . . . . . . . . . . . . . . . . . . 704FROM KINETIC ISOTOPE

EFFECTS TOTRANSITION-STATESTRUCTURE . . . . . . . . . . . . . . . . . . . . 706Matching Kinetic Isotope Effects to

Transition-State Models . . . . . . . . 706Electrostatic Potential Maps . . . . . . . . 706

DESIGN OF TRANSITION-STATEANALOGS . . . . . . . . . . . . . . . . . . . . . . . 706Predictive Binding . . . . . . . . . . . . . . . . . 707Selecting Targets . . . . . . . . . . . . . . . . . . 707

TRANSITION-STATESTRUCTURES ANDANALOGS . . . . . . . . . . . . . . . . . . . . . . . 708Human and Bovine Purine

Nucleoside Phosphorylases . . . . . . 708Human 5′-Methylthioadenosine

Phosphorylase . . . . . . . . . . . . . . . . . . 711Bacterial Methylthioadenosine/

AdenosylhomocysteineNucleosidase Hydrolases . . . . . . . . 712

Ricin A Chain and Saporin . . . . . . . . . 717Orotate

Phosphoribosyltransferases . . . . . . 718GENERAL APPLICABILITY OF

ENZYMATIC TRANSITION-STATE ANALYSIS . . . . . . . . . . . . . . . 718

TRANSITION-STATE VERSUSGROUND-STATECOMPLEXES . . . . . . . . . . . . . . . . . . . . 720Purine Nucleoside Phosphorylase

Transition-State Motion andLifetime . . . . . . . . . . . . . . . . . . . . . . . . 720

Test of the Reactive IntermediateHypothesis for Human PurineNucleoside Phosphorylase . . . . . . . 721

Thermodynamics of Transition-State Analog Binding. . . . . . . . . . . . 722

SUBSTRATE AND TRANSITION-STATE BINDING ISOTOPEEFFECTS . . . . . . . . . . . . . . . . . . . . . . . . 725

CONCLUSIONS ANDTHE FUTURE . . . . . . . . . . . . . . . . . . . 726

INTRODUCTION

Research in the mechanisms of enzymaticcatalysis has redoubled with new efforts incomputational chemistry proposing expla-nations for catalytic rate enhancement andbarrier crossing (1–5), in systems biology forannotation of unassigned open reading frames(6, 7), in chemical library screening for newenzyme inhibitors as pharmacology leads (8,9), in the evolution of enzyme structure andfunction (10, 11), and in the de novo design ofenzymatic activity (12, 13). Catalytic efficiencyand the nature of enzymatic transition statesare pertinent to each effort. Only by efficientformation of the transition state can enzymesserve their catalytic function. Any interferencein catalytic function constitutes an approachto potential pharmacophores. Enzymes re-main important pharmaceutical targets asapproximately one-third of current drugs actas enzyme inhibitors (14, 15). All biologicalfunction is linked to catalytic activity, andincreased understanding in genetics invariablygenerates new questions relative to the catalystsinvolved in higher-order regulatory processes.

Chemical transition states have short life-times of a few femtoseconds, the time requiredfor electron redistribution and the time to con-vert the restoring mode of a chemical bond toa translational mode (16, 17). Enzymes havefunctional turnover numbers (kcat) near the mil-lisecond timescale; thus, the transition-statelifetime occupies only about 10−12 of the re-action coordinate cycle (Figure 1). An en-zyme fully saturated with reactants and en-gaged in maximal catalysis will therefore haveabout 1 part in a trillion occupied by thetransition-state complex with the remainder inground-state complexes. Thus, spectroscopicor static techniques are incapable of charac-terizing enzymatic transition-state complexes.Although claims have been made for trap-ping actual transition-state species in crystallo-graphic structures (e.g., References 18 and 19),these claims have not withstood critical analysis(20, 21).

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1

Tim

e

ms

μs

ns

ps

s

fs

kcat PNP

Bond vibration; TS lifetime

Collisonless H+ ion transfer

Rate O2 Hb to deoxy Hb

Flap opening rate TIM

H2O diffuses two diameters

1017

104

102

10–2

10–4

10–6

10–8

10–10

10–12

10–14

Age of the universe

Flap closing OPRTase

Rotation of amino acid groups

Domain motions in proteins

Light travels 0.3 mm

h Dissociation rate for TS analogs

Common enzymatic kcat range

Billionsof years

min Slow-onset tight binding

Figure 1Relative timescales for motions involved inenzymatic catalysis and interactions of transition-state analogs (adapted from Reference 51 withpermission). Note the 1013 timescale differencebetween kcat for PNP and TS lifetimes (blue). Thered bar indicates typical enzymatic kcat ranges. fs,femtosecond; Hb, hemoglobin; μs, microsecond;ms, microsecond; ns, nanosecond; PNP, purinenucleoside phosphorylase; ps, picosecond; TIM,triose phosphate isomerase; TS, transition state.

Kinetic isotopeeffect (KIE):a ratio of reaction ratesfor labeled (klabel) andunlabeled reactants(kunlabeled); KIE =(kunlabeled)/(klabel)

Binding isotopeeffect (BIE): a ratioof dissociationconstants for labeledand unlabeled ligands;BIE = (Kdunlabeled)/(Kdlabel)

Transition-stateanalysis:(a) measuring intrinsicKIEs for atomiccenters and(b) matching acomputationaltransition state to KIEs

Indirect methods used to characterizetransition states have traditionally beenborrowed from physical organic chemistry,including chemical reactivity relationships(22, 23). Kinetic isotope theory and kineticisotope effects (KIEs) were first developed forisotope fractionation and later used to deducechemical mechanisms in organic chemistryand transition-state features for solution-basedchemical reactions (24–27). The application ofisotope effects to enzymatic reactions becamemore practical with the publication of books

summarizing the theory and methods forapproaching problems with isotope effectsin enzymology (28, 29). With the currentapplications of KIE experiments exemplifiedhere, technology is now available to understandthe structural features of enzymatic transitionstates.

Enzymes are remarkable for catalytic rateenhancement and for their ability to overcomethe single large transition-state energetic bar-rier of solution reactions by creating multiplesteps with smaller barriers. Often, protein con-formational changes or rates of reactant releaselimit the reaction rates and thereby obscure thevalues of the KIE (intrinsic values) arising fromthe chemical step. Intrinsic isotope effects areessential for interpretation of KIE informationinto transition-state structures, and methods toaddress this problem evolved with the applica-tion of transition-state theory to enzymes (30,31). Multiple KIE approaches were used to mapgeneral features of enzymatic transition statesbefore computational approaches were avail-able to fit a family of intrinsic KIEs to a spe-cific transition-state structure (32). Despite thelong history of KIE applications to enzymaticreactions, the technology is still evolving. Forexample, a general assumption has been thatbinding isotope effects (BIEs) could be ignored.BIEs are now known to be significant in many,and possibly all, enzymatic reactions and oftencontribute to the calculated intrinsic KIE val-ues (33, 34). BIEs can now be used to provideinsights about ground-state (thermodynamic)interactions.

This article provides some examples ofthe combined experimental-computationalapproaches to establish transition-state infor-mation for specific enzymatic reactions andthe use of transition-state structure to designtransition-state analogs. The focus is on transi-tion states for a family of N-ribosyltransferases.Despite this focus, the KIE method oftransition-state analysis is general and reliesonly on the ingenuity of the investigator toselect appropriate targets, resolve featuresof the transition state, and synthesize newtransition-state analogs. Isotope effects and

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Molecularelectrostaticpotential: electronforce applied to apositive charge in adefined positionrelative to a molecule

transition-state analogs also provide researchtools to understand catalyst function.

FROM KINETIC ISOTOPEEFFECTS TOTRANSITION-STATESTRUCTURE

KIE measurements compare reaction rates ofisotope-labeled and natural abundance reac-tants. When the reactants compete, the resultsyield KIEs on kcat/Km and include all steps be-tween reactants free in solution and the firstirreversible step in the reaction. These meth-ods have been described (35–38). Recent appli-cation of NMR methods allows natural abun-dance determination of many isotope effectssimultaneously or permit accurate analysis byNMR neighbor chemical-shift methods (39–41). Larger isotope effects from solvent 2H2Oor for hydride transfer mechanisms can be mea-sured using labeled and unlabeled reactants inseparate experiments to obtain distinct valuesfor isotope effects on kcat and Km. Intrinsic KIEsarise from the difference in bond vibrationalenvironment for an atom in the reactant statecompared to its environment at the transitionstate and thus gives an atom-by-atom descrip-tion of the transition state. Multiple isotope ef-fects, taken from molecular sites that change inbond vibrational environments between reac-tant and transition states, can be used to recre-ate a full model of the transition state.

Matching Kinetic Isotope Effects toTransition-State Models

Atom-by-atom KIE values are converted to aspecific static model with fixed bond angles andlengths by computational matching to a quan-tum chemical model of the reaction of inter-est. A typical approach is to explore the reac-tion of interest in the Gaussian computationalsuite using the B3LYP method with a 6-31G∗

basis set (defining the level of computationaltheory), although other levels of theory can beused and the method is not strongly depen-dent on the basis set (42–44). Reactants, transi-

tion state, and product geometries are locatedas the global minima. Transition-state struc-tures are located with a single imaginary fre-quency, characteristic of true potential energysaddle points. The experimental intrinsic KIEsare then matched in fixed-distance optimiza-tions using a grid of possible transition struc-tures. Fixed bonds are used only along the re-action coordinate (the reaction coordinate isdefined as the sites of bond making and bondbreaking, for example, the bonds to the leav-ing group and to the attacking nucleophile inribosyl transferases). The computed structuresare relaxed in other dimensions, and isotope ef-fects are calculated for each transition state us-ing QUIVER or ISOEFF98 software (45, 46).If indicated by structural data and if needed toobtain a KIE match, hydrogen bonds or otherinteractions from catalytic site residues can beadded. Transition-state models matching theintrinsic KIE can be recalculated at the highestpractical level (typically, B3LYP/6-31+G∗∗) toobtain the final transition-state model.

Electrostatic Potential Maps

The CUBE subprogram from Gaussian can beused to generate the molecular electrostatic po-tential surfaces of transition-state structures us-ing the formatted checkpoint files from geom-etry optimization. The molecular electrostaticpotential surfaces can be visualized at the vander Waals surface or closer to the nuclei (iso-values of 0.040 and 0.068, respectively) usingGaussView 3.09 (available in Reference 47).

DESIGN OF TRANSITION-STATEANALOGS

The goal of transition-state analog design isto create stable chemical structures with vander Waals geometry and molecular electrostaticpotential surfaces as close as possible to thoseof the transition state. The original hypothe-sis for binding of transition-state analogs in-voked tight binding of the transition state asthe rationale for the tight binding of transition-state mimics (48–50). This interpretation is now

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Purine nucleosidephosphorylase(PNP): catalyzes thephosphorolysis of6-oxypurinenucleosides and6-oxypurine2′-deoxynucleosides topurine bases andα-D-ribose1-phosphate or2-deoxy-α-D-ribose1-phosphate

Immucillin:a chemically stableanalog of the transitionstates for 6-oxypurineN-ribosyltransferaseswith early transitionstates

debated as the explanation for transition-state formation (discussed below). A dynamicview proposes that enzyme-associated reactantscross the transition-state barrier by stochasticprotein vibrational modes without unique equi-librated high-affinity binding features (51, 52).Transition-state analogs convert the dynamicfeatures of the transition state into thermo-dynamic binding energy and thereby generatetight binding.

Predictive Binding

Before synthetic chemistry efforts are appliedto putative transition-state analogs, quantita-tive measures of similarity to the transition stateare useful. One method ranks the similarityof proposed analogs by parameters of geom-etry and electrostatic potential surface againstthe same parameters for transition-state analogs(53, 54). As geometry and electrostatics are thedominant parameters for ligand recognition,molecules more similar to the transition stateare bound more tightly. For example, usingthese similarity parameters from 0 (no similar-ity) to 1.0 (identity to the transition state), thesubstrate inosine for bovine purine nucleosidephosphorylase (PNP) shares a 0.483 similar-ity, whereas the analog immucillin-H (ImmH)shares a similarity of 0.723 to the transitionstate, providing a dissociation constant of 23picomolar (pM) and a Km/Kd ratio of 739,000(Figure 2) (55). Values for chemically stabletransition-state analogs near 1.0 are not possi-ble because the features of the transition stateinclude nonequilibrium bond lengths and par-tial charges that cannot be reproduced in stablechemical mimics.

A second predictive affinity method usesneural network similarity measures to rankgeometry and electrostatic potential surfaceparameters from a known set of inhibitors(including some with features of the transitionstate) and compare these parameters to thosefrom proposed inhibitors prior to chemicalsynthetic efforts (56). This approach faithfullypredicts the binding affinity of a family of un-

–10

–15

–20

–25

–30

–35

–40

–450.4 0.5 0.6 0.7 0.8 0.9 1.0

Inosine

Immucillin-H

Transition state

ΔG/RT

Se

Figure 2Binding free energy (�G/RT ) and molecular electrostatic potential surfacesimilarity 〈Se〉 for inosine, immucillin-H, and the transition state for bovinepurine nucleoside phosphorylase (modified and reprinted from Reference 55with permission).

known inhibitors for the IU-nucleoside hydro-lase from Crithidia fasciculata (Figure 3) (57).

Selecting Targets

Emphasis on the N-ribosyltransferases in thisreview is not a random choice. Good targetcandidates for transition-state analog designshould have altered geometry and/or chargeon conversion from reactants to the transitionstate. Glycosytransferases generally share theseproperties. Thus, most sugar transferases formtransition states with cationic charge at theanomeric carbon, and the geometry is alteredat this center from sp3 (tetrahedral geometry)in the reactant sugar to sp2 (trigonal planargeometry) at the transition state (58–60). Thus,the chemistry to match these features providescandidates as transition-state analogs (e.g., Ref-erences 61 and 62). Other reactions that involvedisplacements at carbon are also candidates fortransition-state analysis. Such reactions includea broad range of biological reactions with al-tered charge and/or geometry at the transitionstate, including reactions with sp2 reactants andsp3 transition states, for example, the proteases.


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Transition state

High-affinity inhibitor

Low-affinity inhibitor

SurfacePartial

positivecharge

Partialnegativecharge

Relativecharge

Figure 3A molecular electrostatic potential and geometric input for inhibitor recognition by nucleoside hydrolaseusing a neural training network. The surface (outer sphere of golden squares) is imprinted with molecularelectrostatic potentials, where red indicates a partial positive charge, and purple indicates a partial negativecharge relative to the average charge at the van der Waals surface. The transition state (left) is similar to thehigh-affinity inhibitor (middle, Kd = 4 nM) but different from the low-affinity inhibitor (right, Kd =18 mM). Modified from Reference 57 and reprinted with permission. nM, nanomolar; mM, millimolar.

TRANSITION-STATESTRUCTURES AND ANALOGS

Examples of transition-state analysis are pro-vided where transition-state information hasbeen obtained from KIEs and computationalchemistry. Transition-state analogs have beendesigned, synthesized, and characterized formost of these targets. Bovine and human PNPsare the most advanced examples, and candidatesfrom the first and second generation transition-state analog inhibitors are currently in advancedclinical trials (see below). Other examples pro-vided here have yielded the most powerful in-hibitors known for the targets.

Human and Bovine PurineNucleoside Phosphorylases

The human genetic deficiency of PNP waslinked to T-cell immune deficiency by theearly studies of Giblett et al. (63). Subsequentstudies revealed that the accumulation of 2′-deoxyguanosine in blood is specifically toxic bycausing excess dGTP accumulation in activatedT cells (64). Bovine PNP was used as a modelfor human PNP and assumed to form a simi-lar transition state because of the 87% aminoacid sequence identity (65, 66). KIE values andtransition-state analysis for the arsenolysis re-action revealed an early dissociative transition-state structure with a significant bond order to

the departing hypoxanthine at the transitionstate (Figure 4). Electrostatic comparison ofthe transition state and analogs indicated a goodmatch between ImmH and the transition statefor bovine PNPs, and indeed, ImmH is a 23-pMinhibitor of bovine PNP and gives a 739,000Km/Ki value (Figures 3 and 4). As bovine PNPwas intended as a surrogate for human PNP,the 56-pM dissociation constant of ImmH forhuman PNP was a concern because of its loweraffinity for the desired target, especially becausethere is 100% amino acid conservation for in-hibitor contacts at the catalytic sites (67). Thisdifference in ImmH binding for the human andbovine PNP isozymes suggested the possibilityof distinct transition states.

Intrinsic KIE values for bovine and humanPNPs were different (Figure 4). Becauseintrinsic KIEs report on the bond vibrationalstatus at the transition state, the primarydata set established different transition-statestructures for these closely related enzymes(Figure 4). The transition state for humanPNP is a fully formed ribocation with thecationic charge located primarily at C1′ (theanomeric carbon), has a fully dissociatedbond of 3 A to the hypoxanthine, and showsno significant participation of the arsenatenucleophile. The physiological substrate forhuman PNP is 2′-deoxyguanosine; thus, thetransition state for human PNP also forms

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O4' O4'C5' C5'

3H5' 3H5'3H5'

O

N7

14C 14C

HO OH HO OH

N9

N

NH

HO3H1' 3H1'

3H3' 3H3'3H2' 3H2'

3H4' 3H4'14.1

3H5'

3.3

15.2

1.0

2.6

0.8

O

N7

N9

N

NH

HO

18.4

6.2

3.1

2.9

0.2

2.4

N

N

N

HN

O

O

OH

HO

OH

As OHO

O

–O

H

Bovine PNPtransition state

‡

3.0 Å

1.77 Å

δ+

N

N

N

HN

O

O

OH

HO

OH

As OHO

O

–O

H

Human PNPtransition state

‡

3 Å

3 Å

δ+

Bovine PNP Human PNPIn

trin

sic

kin

eti

c is

oto

pe

eff

ect

s R

ea

ctio

n c

oo

rdin

ate

b

on

d l

en

gth

s M

ole

cula

r e

lect

rost

ati

c p

ote

nti

als

TS ImmH

23 pMTS DADMe-ImmH

9 pM

Partialnegativecharge

Partialpositivecharge

Figure 4Intrinsic kinetic isotope effects (in percentages) for bovine (upper left, in red ) and human (upper right) purinenucleoside phosphorylases (PNPs). Intrinsic isotope effects shown in blue for human PNP are significantlydifferent from those for bovine PNP. Reaction coordinate bond lengths (middle) give rise to the molecularelectrostatic potentials (bottom) for the transition states (TSs) and transition-state analogs, immucillin-H(ImmH), and 4′-deaza-1′-aza-2′-deoxy-1′-(9-methylene)immucillin-H (DADMe-ImmH). Blue atoms(middle) are sties most changed by the formation of the transition state. pM, picomolar.

efficiently with 2′-deoxyribosyl sugars. Thesedistinct features of the transition state can bematched with DADMe-ImmH [4′-deaza-1′-aza-2′-deoxy-1′-(9-methylene)immucillin-H](Figures 4 and 5). DADMe-ImmH showshigher affinity for human than bovine PNP and

binds 2,400,000 times tighter than substrateaccording to the Km/Kd ratio (68).

Lessons from comparisons of bovine andhuman PNPs are that distinct transition-statestructures can be formed by closely relatedenzymes with nearly identical catalytic sites


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HN

NNH

H2N

O

HO

OH OH

HN

N

HN

NH

O

HO

OH

HN

N

HN

NH 2HO

O

HOOH

HN

N

HN

NH 2

O

HO

HO

ImmH56 pM

SerMe-ImmH5 pM

DATMe-ImmH9 pM

DADMe-ImmH9 pM

Figure 5Four generations of transition-state analogs for human purine nucleosidephosphorylases together with their dissociation constants. DADMe-ImmH,4′-deaza-1′-aza-2′-deoxy-1′-(9-methylene)immucillin-H; ImmH, immucillin-H; pM, picomolar; SerMe-ImmH, the seramide substituent of9-methylene-9-deazahypoxanthine.

and that transition-state information fromKIEs provides a tool for the design of spe-cific transition-state analogs. Distinct transi-tion states from enzymes with near-identicalcatalytic sites suggest involvement of the pro-tein architecture in transition-state formation.By replacing remote amino acids (>25 A fromthe catalytic sites) in human PNP with thoseof bovine PNP, a chimeric enzyme was formedwith altered dynamic properties and a transitionstate distinct from either parent (69, 70).

ImmH contains four asymmetric carboncenters and reactive nitrogen and oxygengroups that require protection, making thechemical synthesis difficult (71, 72). Use of2′-deoxynucleosides by human PNP permitsdesign of transition-state analogs with the elim-ination of one stereocenter, and replacing theC1′-anomeric carbon with nitrogen eliminatesa second; thus, DADMe-ImmH has only twostereocenters and can be synthesized usingthe Mannich reaction without protecting thechemically reactive nitrogen and oxygen atoms(Figure 5) (73). Replacing the hydroxypyrroli-dine ribocation mimic of DADMe-ImmH withacyclic groups further simplified these analogsand has generated third- and fourth-generationinhibitors exemplified by DATMe-ImmH andSerMe-ImmH (Figure 5). These inhibitorsshow similar picomolar binding affinity to hu-man PNP and are increasingly accessible to syn-thetic chemistry (74, 75). Crystallographic anal-

ysis of human PNP with all four generations ofthe immucillins shows common features linkedto their tight binding. Most important is theability to form an ion pair between the boundN-ribocation mimic and the phosphate anion atthe catalytic site. In ImmH, this distance is 3.3 Aand is shorter than with the other analogs (67).The 9-deazahypoxanthine group is common toall inhibitors, but in ImmH, contacts to catalyticsite groups are less optimal than for the oth-ers. The geometry of ImmH in the dimensionof the reaction coordinate (distance from theN4′-cation to N7) is too short to permit opti-mal contacts to both 9-deazahypoxanthine andthe ribocation mimic. Thus, human PNP is re-sistant to flex beyond the extended geometry ofa fully dissociated transition state in this dimen-sion. Inhibitors longer in their reaction coordi-nate dimension by addition of the methylenebridge more optimally capture the interactionsbetween the inhibitor and the catalytic site.

Transition-state analogs are not useful indrug development unless biological availabil-ity permits access to the target. In the caseof human PNP, this means access to all tis-sues because the enzyme is widely distributed.From characterization of human PNP geneticmutants, it is known that >95% inhibition ofPNP is needed to cause acumulation of 2′-deoxyguanosine and thereby block T-cell pro-liferation (76). ImmH enters human cells onthe purine nucleoside transporters to provideaccess to all tissues (77).

Although mechanisms of transport for otherimmucillins have not been reported, single-dose oral administration of DADMe-ImmHcaused rapid inhibition of PNP in mice as indi-cated by complete inhibition of PNP in mouseblood. Recovery of half-normal PNP activityrequires 11.5 days and parallels erythrocyte re-placement. Thus, DADMe-ImmH stays on itsbiological target until cells are replaced (68).Imm-H and DADMe-ImmH show low toxic-ity in animal studies. ImmH is in clinical tri-als for T-cell malignancies under the name ofForodesineTM, and DADMe-ImmH is in clin-ical trials for gout under the name BCX4208(78, 79).

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H2NH2N

H2N

H2N H2N

H2N

NH2

COOH

Me

S+

Me

MeS

Methionine

SAM

ATP

PRPP

MeS

S+

HO OH HO OH

HO OH

HO OH

OPO3

N

N

N

NN

N

NNO

O

O

MTA

Spermidinesynthase2.5.1.16

Spermidine Spermine

Sperminesynthase2.5.1.22

Decarboxy-SAM

Decarboxy-SAM

Putrescine

OrnithineOrnithine decarboxylase

Methylations

MTAPN

N

NN

N

N

NNH

NH

NH

NH

OCOOH

NH2

NH2NH2

NH2NH2

NH2

NH2

Figure 6The role of 5-methylthioadenosine phosphorylase (MTAP) in polyamine synthesis, methionine, ATP, andS-adenosylmethionine (SAM) salvage (figure modified and reprinted from Reference 87 with permission).

5′-Methylthioadenosinephosphorylase(MTAP): an enzymethat converts 5′-methylthioadenosine(MTA) and phosphateto adenine and5-methylthio-α-D-ribose1-phosphate

Human 5′-MethylthioadenosinePhosphorylaseThe production of 5′-methylthioadenosine(MTA) in humans occurs only in the pathwayfor conversion of S-adenosylmethionine (SAM)to polyamines (Figure 6). The sole metabolicfate for MTA is conversion to adenine and5-methylthio-α-D-ribose 1-phosphate by 5′-methylthioadenosine phosphorylase (MTAP).These products of the MTAP reaction are re-cycled to ATP and methionine, metabolites thatcan be recycled to SAM (80). MTAP thereforeserves as an essential part of adenine, methio-nine, and SAM salvage pathways. The MTAPreaction is linked to biosynthetic methylationreactions and regulatory methylations of CpGsites in DNA and to protein methylation. In-hibitors of these processes are proposed tofunction as anticancer agents (81, 82). Thepolyamine pathway is also an anticancer target.

Inhibition of MTAP causes MTA to accumu-late and causes product inhibition of polyaminesynthetases (82, 83).

KIEs for human MTAP and computationalmodeling (see above) provided a transition-state structure for the arsenolysis of MTA(84). Intrinsic KIEs indicated a dissociative SN1transition state for MTAP (Figure 7). Theanomeric carbon of 5-methylthioribose is con-verted to a cationic center at the transition state,and bond loss to the adenine leaving group iscomplete. An unusual feature of the KIEs isthe large [9-15N]MTA value of 3.9%, which in-dicates anionic adenine at the transition state.The primary [1′-14C]MTA KIE of 3.1% indi-cates a transition state with significant bond or-der between the anomeric carbon and the at-tacking nucleophile, an arsenate oxygen (2.0 Adistant from the anomeric carbon). Anotherunusual feature of this transition state is the


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1.086±0.002 1.046±0.001NH2

NH2 ‡NH2

NH2 NH2

4-Cl-PhS

NH2

N

N NN

N

N NN

HO

HO

5'-Methylthio-adenosine

Km = 5 μM

Ki* = 1,000 pM

MT-immucillin-A MT-DADMe-immucillin-A pCIPhT-DADMe-immucillin-AKi* = 86 pM Ki* = 10 pM

Michaelis complex MTAP transition state

HOHO OH

MeSMeS

N

NH+

NH+

+

HNH

NH

NH2

NN

N

O

N

N

N

O

O– O–

O–

OHAsOH

δ+

δ–

MeS

MeS

O

O–

O–

OH

OH

AsO

N

N

N15N

14C3H3H

3H3H3H3C

SO

3H

HO OH

1.037±0.0021.030±0.005

1.350±0.0031.076±0.003

1.045±0.005

Figure 7The transition state and transition-state analogs for the arsenolysis reaction of human5′-methylthioadenosine phosphorylase (MTAP). The intrinsic kinetic isotope effects used in transition-stateanalysis are shown on the upper right, and the inhibitors are shown below. Inhibitor features that mimic thetransition state are in blue. pClPhT- is a 4-chloro-phenylthio group. MT, methylthio; pM, picomolar.

polarization or ionization of the ribosyl 3′-OHgroup. Ionization of the 3′-OH group suggestsa zwitterionic ribosyl group with a 3′-endo con-formation at the transition state.

The late SN1 transition state for hu-man MTAP suggested that analogs similarto DADMe-ImmH for human PNP, mod-ified to reflect the substrate specificity ofhuman MTAP, may capture transition-statefeatures. The 9-deazaadenine ring and 5′-methylthio group mimic human MTAP sub-strates (85). Analogs for the human MTAPtransition state were synthesized and pro-vide picomolar inhibitors (84–86). Methylthio-DADMe-immucillin-A (MT-DADMe-ImmA)is a transition-state analog of human MTAP andis a slow-onset, tightly binding inhibitor witha dissociation constant of 86 pM and is orallyavailable in mice (Figure 7) (87).

Screening of human cancer cell lines showedlow toxicity for MT-DADMe-ImmA and MTAas individual agents. MT-DADMe-ImmA incombination with MTA causes growth inhibi-

tion in some cell lines and apoptosis in oth-ers. Human FaDu head and neck cancer cells,when treated with MT-DADMe-ImmA andMTA, revealed inhibition of cellular MTAP,increased cellular MTA concentrations, de-creased polyamines, and increased rates ofapoptosis. Implanted FaDu tumors in mousexenografts were treated with MT-DADMe-ImmA, resulting in tumor remission with noapparent toxicity to the host (87). In a re-lated study with human lung cancer cell lines,MT-DADMe-ImmA inhibited growth of pri-mary tumors in mice and decreased metastaticcancers to the lungs (88).

Bacterial Methylthioadenosine/Adenosylhomocysteine NucleosidaseHydrolases

Induction of luminescence genes in Vibrio har-veyi as a function of cell density in cultureled to the discovery of quorum-sensing sys-tems in bacteria (89). When bacterial cell

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5′-Methylthioadeno-sine/adenosylhomo-cysteine nucleosidase(MTAN): an enzymethat catalyzes thehydrolysis of 5′-methylthioadenosine(MTA) and adenosyl-homocysteine toadenine and 5-methylthio-α-D-ribose orribosylhomocysteine

populations reach critical levels, cells produceand detect signaling molecules known as au-toinducers (AIs) to coordinate gene expressionand regulate processes that are presumed to bebeneficial for the colony (89). Bacterial mul-tidrug resistance has rapidly appeared with con-ventional cell-killing antibiotics, and overcom-ing this problem requires approaches that arenonlethal to bacteria to prevent development

of resistance while blocking pathogenicity (90–93). Quorum sensing is linked to virulence butis not essential for growth, making it an attrac-tive target.

One class of quorum-sensing molecules,the autoinducer-1 (AI-1) and autoinducer-2(AI-2) molecules, is linked to SAM metabolism(Figure 8). 5′-Methylthioadenosine/adenosyl-homocysteine nucleosidases (MTANs)

N

NN

N

NH2

NH2

NH2NH2

O

HO OH

SHOOC

H2N

H2N

H2N

H2N

N

NN

NO

HO OH

SHOOCN

NN

NO

HO OH

S

SO

HO OH

SHOOCO

HO OH

O

NH

R

O

O

OHOH

SHHOOCS COOH

O

OB

O

HO OH

Acyl-ACP

AHLsynthaseHomoserine

lactones (AI-1)

SAM biosynthesis

Putrescine

CO2 +polyamines

Methionine

Methyltransferasereactions

Adenine

SAM

MTA

H2OH2O

MTR

Adenine

THF CH3-THF

SRH

SAH

HomocysteineMetH

MTAN

Tetrahydrofurans (AI-2)

MetE

RH-cleavage enzyme

Figure 8The role of 5′-methylthioadenosine/adenosylhomocysteine nucleosidase (MTAN) in AI-1 and AI-2quorum-sensing pathways (reprinted from Reference 94 with permission). AHL, acylhomoserine lactones;AI, autoinducer; MTA, 5′-methylthioadenosine; MTR, methylthioribose; SAH, S-adenosylhomocysteine;SAM, S-adenosylmethionine; SRH, S-ribosylhomocysteine.


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hydrolyze both MTA and adenosylhomocys-teine, are directly involved in the biosynthesisof autoinducers or in SAM recycling, and areessential for the production of autoinducermolecules (94). AI-1 and AI-2 are synthesizedfrom SAM; thus, MTAN inhibition was pro-posed to block both AI-1 and AI-2 production,thereby disrupting quorum sensing. BecauseMTANs are not found in humans, MTANinhibitors are expected to block quorumsensing in bacteria without effects on humanmetabolism.

Transition-state structures of bacterialMTANs were established by quantum chem-ical analysis using the intrinsic KIE valuesfor MTANs from Escherichia coli, Streptococ-cus pneumoniae, and Neisseria meningitidis (95–97). A comparison of the intrinsic KIE val-ues is instructive (Figure 9). Intrinsic KIEsfor E. coli MTAN-catalyzed hydrolysis of MTAgave large 1′-3H (16%) and small 1′-14C (0.4%)KIEs, indicating that this transition state in-volves minimal leaving group or attacking nu-cleophile participation and a transition statewith well-developed ribocation character. Atransition state matching the intrinsic KIEs waslocated and indicated the leaving group (N9)was 3.0 A from the anomeric carbon and a simi-lar distance for the attacking water nucleophile.The relatively small 9-15N KIE indicates that

N. meningitidis 1.9E. coli 1.8S. pneumoniae 3.7

3.0

NH2

N

N15N

1623

3.20.40.0

–1.24.49.5

3H3HC

14CO

N

S

3H

3H

3H

3H

3H

3H

HO OH

Figure 9Kinetic isotope effect values (as percentages) usedfor transition-state analysis of the bacterial5′-methylthioadenosine/adenosylhomocysteinehydrolases.

the leaving group is protonated at the the tran-sition state. Ribose pucker at the transition stateaffects the 2′-3H KIE, and the relatively smallvalue of 4.4% indicated a H1′-C1′-C2′-H2′ di-hedral angle of 53◦ at the transition state, amodest 3′-endo geometry. This transition statepredicts that extended transition-state analogs,patterned after the DADMe-ImmH for humanPNP, would resemble this transition state, andthese compounds are powerful inhibitors (seebelow).

The intrinsic KIE values for S. pneumoniaeMTAN are similar to those for the E. coli en-zyme and also support a dissociative SN1-liketransition state with no significant covalentparticipation of the adenine leaving groupor the attacking water nucleophile (96). Aquantum chemical model of the transition stateas a ribooxacarbenium ion intermediate wasfound to fit the intrinsic KIEs. A 3′-endo con-formation for the ribocation corresponding toH1′-C1′-C2′-H2′ dihedral angle of 70◦ is con-sistent with the KIEs. Although both E. coli andS. pneumoniae MTAN transition states exhibitfully developed ribocations, the 9-15N KIEsdiffer considerably, 1.8% and 3.7%, respec-tively. The [9-15N]MTA isotope effect reportson the total bond order to N9 at the transitionstate and is influenced by the protonation stateof the leaving group. The value of 3.7% foundfor the S. pneumoniae MTAN indicates that theadenine leaving group is not protonated at thetransition state and therefore is proposed to de-part as a catalytic-site-stabilized adenine anion.In this and other transition states, the influenceof the virtual solvent (a dielectric constant)was varied as part of the modeling and did notinfluence calculated KIE values beyond exper-imental error. S. pneumoniae MTAN differsfrom most other purine N-ribosyltransferasesbut resembles human MTAP in the protona-tion state of the leaving group. Departure of aleaving group from an ion-pair transition state(cationic ribose and anionic adenine) is difficultand is consistent with the 103-fold decrease inthe catalytic efficiency of S. pneumoniae MTANrelative to that from E. coli. Because the interac-tion of transition-state analogs is also related to

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catalytic efficiency, this transition state is alsoconsistent with weaker binding of transition-state analogs to S. pneumoniae than to E. coliMTANs (see below).

Unlike the well-developed ribocation tran-sition states of S. pneumoniae and E. coliMTANs, the transition state of N. meningitidisMTAN is early in an SN1 reaction path. The 1′-3H KIE is dominated by an out-of-plane modeas the anomeric carbon rehybridizes from sp3

in the reactant to sp2 in dissociative transitionstates. In E. coli and S. pneumoniae MTANs,the 1′-3H KIEs are 16% and 23%, but for N.meningitidis MTAN, the value is 3%. Thus,sp2 geometry at C1′- is not established at the

transition state. Likewise, no isotope effect isseen at 2′-3H, and the isotope effect of 9-15N is1.9%. These values are consistent with the sig-nificant bond order remaining to the adenineleaving group at the transition state. S. pneu-moniae MTAN, like bovine PNP, is the sec-ond enzyme in the purine N-ribosyltransferasefamily to exhibit an early transition state. Latedissociative SN1 transition states dominate theN-ribosyltransferases.

Transition-state analogs designed forhuman MTAP overlap in activity with thosedesigned for bacterial MTANs (Figures 7and 10). Iminoribitol analogs of MTA mimicearly dissociative transition states for MTAN.

NH2

NH2 NH2 NH2

NH2NH2

FNH2NH2

N

N

NH2

NH2

N

N

N

N

S SS

S S S

S S

Cl

Cl

HO

N

N

N

N

N

S

S S S O

HO

HO HO HO HO HO

pClPhT-DADMe-ImmAKi* = 47 fM

PrT-DADMe-ImmAKi* = 580 fM

4-PyrT-DADMe-ImmAKi* = 2 pM

MT-DADMe-ImmAKi* = 2 pM

5'-dEt-DADMe-ImmAKi* = 6 pM

Homocys-DADMe-ImmAKi* = 6 pM

BnO-DADMe-ImmAKi* = 9.0 pM

cHxT-DADMe-ImmAKi* = 740 fM

mClPhT-DADMe-ImmAKi* = 742 fM

EtT-DADMe-ImmAKi* = 950 fM

PhT-DADMe-ImmAKi* = 2 pM

BuT-DADMe-ImmAKi* = 296 fM

BnT-Pz-DADMe-ImmAKi* = 400 fM

BnT-DADMe-ImmAKi* = 460 fM

pFPhT-DADMe-ImmAKi* = 550 fM

HO

S

HO HO HO HO

HO HO HO

H

NH

NH NH2

NH2

COOH

N

N

N

NH NH2

N

N

N

NH NH2

N

N

N

NH NH2

N

N

N

NH

N

N

NH2

N

N

N

N

N N

H

NH

N

N

N

N

NN

N

H

NH

N

N

N

NH

N

N

N

NH

N

N

N

N

H

N

N

N

N

H

Figure 10Transitions state analogs of E. coli 5′-methylthioadenosine/adenosylhomocysteine hydrolase. Dissociation constants are shown, and Ki

∗indicates the observation of slow-onset, tight-binding inhibition. The prefix indicates the 5′-substituents to the parent DADMe-ImmA,thus, BnO-DADMe-ImmA, benzyl-O-DADMe-ImmA; BnT-DADMe-ImmA, benzylthio-DADMe-ImmA; BnT-Pz-DADMe-ImmA,benzylthio-pyrazolo-DADMe-ImmA; BuT-DADMe-ImmA, butylthio-DADMe-ImmA; cHxT-DADMe-ImmA, cyclohexylthio-DADMe-ImmA; EtT-DADMe-ImmA, ethylthio-DADMe-ImmA; fM, femtomolar; Homocys-DADMe-ImmA, homocysteinyl-DADMe-ImmA; mClPhT-DADMe-ImmA, meta-chlorophenylthio-DADMe-ImmA; MT-DADMe-ImmA, methylthio-DADMe-immucillin-A; pClPhT-DADMe-ImmA, para-chlorol-phenylthio-DADMe-immucillin-A; pFPhT-DADMe-ImmA, para-fluoro-phenylthio-DADMe-ImmA; PhT-DADMe-ImmA, phenylthio-DADMe-ImmA; PrT-DADMe-ImmA, propylthio-DADMe-ImmA;4-PyrT-DADMe-ImmA, 4-pyridylthio-DADMe-ImmA; 5′-dEt-DADMe-ImmA, 5′-deoxyethyl-DADMe-ImmA.


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0

5

10

15

20

0 200 400 600 800 1000

AI-2

indu

ctio

n (f

old)

BuT-DADMe-ImmA (nM)

IC50 = 1.4 nM

Figure 11Inhibition of autoinducer-2 (AI-2) formation in Vibrio cholerae byBuT-DADMe-ImmA (see Figure 10) (reprinted from Reference 94 withpermission). IC50 refers to the concentration of BuT-DADMe-ImmA thatcauses 50% inhibition of AI-2 formation.

DADMe-immucillin:a chemically stableanalog of the transitionstates for purineN-ribosyltransferaseswith late transitionstates

5′-Methylthio-immucillin-A (MT-ImmA)was a slow-onset tight-binding inhibitorwith a 77-pM dissociation constant forE. coli MTAN, and hydrophobic substituentslowered the dissociation constant to 2 pM. TheDADMe-immucillins are more closely relatedto the transition state, and MT-DADMe-ImmA exhibits a 2-pM dissociation constant.Hydrophobic 5′-substituents in this scaffoldgave transition-state analogs with dissociationconstants in the range of 10−12 to 10−14 M.The dissociation constant of 47 femtomolar(10−15 M) for 5′-p-Cl-phenylthio-DADMe-immucillin-A ( pClPhT-DADMe-ImmA)ranks it among the most powerful noncovalentinhibitors reported for any enzyme. Compar-ison with substrate interactions by the Km/Kd

ratio indicates that this inhibitor binds 91million times tighter than the substrate.

The MTAN inhibitors were tested for theability to disrupt quorum sensing in E. coli andVibrio cholerae. MT-DADMe-ImmA, EtT-DADMe-ImmA, and BuT-DADMe-ImmA(see Figures 10 and 11) bind to V. choleraeMTAN, with dissociation constants of 73, 70,and 208 pM, respectively. Enzymatic assays ofMTAN in intact V. cholerae cells demonstrated

cell permeability and potent MTAN inhibitionwith 50% inhibitory concentration (IC50) val-ues of 6 to 27 nM. To cause quorum-sensingdisruption without induction of resistance,MTAN inhibitors should not disrupt bacterialgrowth. No growth inhibition was observedat concentrations 1,000 times the IC50values,and autoinducer production was blocked atnanomolar concentrations (94). Similar resultswere obtained with enterohemorrhagic E.coli O157:H7. Inhibitor studies conducted inparallel with bacterial cells genetically deletedin MTAN affirmed the role of this pathway inquorum sensing and its potential as a target forbacterial quorum sensing (94).

With the three MTAN transition states de-scribed above, the transition states for othermembers of the bacterial MTAN family can bepredicted from MTAN affinity to transition-state analogs. Thus, transition-state analogswith short distances between the ribocationmimic and leaving group mimic will bind bet-ter to the MTANs that form early transi-tion states. Transition-state analogs with ex-tended distances between the ribocation mimicand leaving group mimic will bind better toMTANs with fully dissociated transition states.This approach was tested on six MTANs, thethree described above with known transitionstates and three MTANs with uncharacterizedtransition states. Using this approach, the tran-sition states of N. meningitidis and Helicobacterpylori MTANs appear similar, and both are earlydissociative. By contrast, E. coli, Staphylococcusaureus, S. pneumoniae, and Klebsiella pneumo-niae MTANs all bind more tightly to extendedtransition-state analogs, resembling fully disso-ciated ribocation transition states. Intrinsic KIEvalues were used to confirm this prediction.Characteristic large [1′-3H]MTA and unity[1′-14C]MTA KIEs were observed for theMTANs predicted to have fully dissociativetransition states, whereas MTANs with earlydissociative states showed small [1′-3H]MTAand significant [1′-14C]MTA KIEs (98). Acaveat to analysis of transition-state struc-ture by transition-state analog specificity is the

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Ribosome-inactivating protein(RIP): an enzymethat catalyzes thefunctional inactivationof ribosomes, oftenwith high specificityand often bydepurination ofadenine

requirement for a family of well-characterizedtransition-state analogs for the enzymes of in-terest, a rarity in the current development oftransition-state analogs.

Ricin A Chain and Saporin

Ricin toxin A chain (RTA) and saporin areplant ribosome-inactivating proteins (RIPs)with catalytic activity for depurination of ade-nine residues from RNA, making them N-ribosylhydrolases (99). Biomedical interest inthe RIPs arises from their use as anticanceragents when linked to monoclonal antibod-ies or other molecules designed to recognizecancer cells. Off-target toxicity causes vascu-lar leak syndrome, an unacceptable side ef-fect. Transition-state analogs that inhibit RIPactivity following therapy are being explored toprovide an approach to limit the side effects.

Ricin A chain shows high substrate speci-ficity for a single adenine in the elongationfactor–binding site of 28S ribosomal RNA lo-cated in a 5′-GAGA-3′ tetraloop where the5′-A is hydrolyzed. The transition state fordepurination of stem-loop RNA by RTA wassolved by KIEs using stem-loop RNA (5′-GGCGAGAGCC-3′) with isotopically labeledadenosine at the depurination site (100). TheKIEs of 16.3% for [1′-3H], −1.9% for [7-15N],1.6% for [9-15N], −0.7% for [1′-14C], and 1.2%for [2′-3H] demonstrated formation of an RNAribooxocarbenium ion and adenine in equilib-rium with the Michaelis complex (101). Theribosyl ring pucker at the transition state wasfound to be 3′-endo, giving rise to the small[2′-3H] KIE. The unusual dihedral angle of ap-proximately 50◦ between C2′-H2′ and the va-cant p orbital of the anomeric carbon are con-sistent with the relatively rigid geometry of thestem-loop RNA backbone (Figure 12). An ir-reversible nonchemical step initiates water at-tack. RTA has a stepwise DN

∗AN mechanism,and the cationic intermediate has a finite life-time. This study with RTA was the first to es-tablish that KIEs and transition-state analysiscould be extended to the reactions of nucleicacid chemistry.

Transition-state analysis for oligonu-cleotides was extended to DNA in a KIE andtransition-state analysis study using RTA withstem-loop DNA as a slow substrate. Althoughthe transition state for stem-loop RNA is con-strained by the rigid RNA scaffold, stem-loopDNA is more flexible and permits formationof the most chemically stabilized geometryfor the deoxyribocation at the transition state(Figure 12) (102).

On the basis of transition-state structures ofRTA with stem-loop RNA and DNA, riboca-tion characteristics with N7-protonated leavinggroups are implicated as transition-state mim-ics. Because RTA is catalytically efficient onlyon stem-loop RNAs, loop mimics of the stem-loop structures were constructed and tested asinhibitors using small stem-loop RNAs as sub-strates (103, 104). Nanomolar inhibitors were

C5'

C1'

O2'

O3'3'-exo

3'-endo

Figure 12Transition-state geometry for ricin A chain withstem-loop RNA (dark gray) and with stem-loopDNA (medium gray) superimposed on the crystalstructure of stem-loop RNA geometry (light gray)(reprinted from Reference 102 with permission).Ribosyl carbons C1′ and C5′ as well as ribosyloxygens O2′ and O3′ are shown for the twogeometries of the transition state, 3′-exo for thetransition state with RNA and 3′-endo for thetransition state with DNA. 3′-Exo and 3′-endoribosyl geometry refer to the 3′C being below orabove the plane of the ribosyl ring formed by theother four atoms.


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achieved with an N-benzyl-hydroxypyrrolidine(N-Bn) transition-state analog at the RTAdepurination site in a circular GAGA motif. Acyclic G(N-Bn)GA provided a new RTA in-hibitory scaffold and the most powerful RTAinhibitor (Kd = 70 nM) known. The transition-state analysis and inhibitor design for RTA wasdone at pH 4.0, required for RTA activity onsmall stem-loop substrates. These inhibitorsdid not protect mammalian ribosomes fromRTA at physiological pH values. Transition-state inhibitor design was therefore extendedto saporin, a RIP from the soapwort plant withthe assumption that it forms a similar transitionstate.

Saporin-L1 from Saponaria officinalis (soap-wort) leaves is highly active against smallnucleic acid substrates and mammalian ri-bosomes and removes multiple adeninesfrom RNA, including small linear sequences(105). Linear, cyclic, and stem-loop oligonu-cleotide inhibitors containing 9-deazaadenine-9-methylene-N-hydroxypyrrolidine and basedon the 5′-GAGA-3′ sarcin-ricin tetraloop gaveKd values in the range of 2 to 9 nM(Figure 13). Under physiological pH values,these analogs bind up to 40,000-fold tighterthan similar RNA substrates. Importantly, thedestruction of ribosomes by saporin-L1 in rab-bit reticulocyte translation was protected bythese inhibitors (106).

The availability of powerful inhibitorsfor ricin and saporin permitted the firstcrystal structures of RTA and saporin witholigonucleotides bound in the catalytic sites.The inhibitors resemble both the sarcin-ricinrecognition sequence and the ribocationtransition state established for RTA. RTAand saporin share unique purine-bindinggeometry at their catalytic sites with quadruplepi-stacking interactions between adjacentadenine and guanine bases and two conservedtyrosines (107). An arginine at one end of thepi stack provides cationic polarization and isproposed to enhance the adenine leaving groupability (Figure 14). Adenine leaving groupactivation dominates the reaction mechanism

with no apparent features to assist in ribocationformation. Conserved glutamates activate thewater nucleophile. Ricin A chain and saporin-L1 exemplify the ability of transition-stateanalogs to organize catalytic sites and permitmechanistically meaningful crystal structures.

Orotate Phosphoribosyltransferases

Orotate phosphoribosyltransferases fromPlasmodium falciparum and human sources(PfOPRT and HsOPRT) use the pyrimidinenucleoside orotidine as a slow substrate forpyrophosphorolysis. Intrinsic KIEs weremeasured to provide the first enzymatictransition state using pyrophosphate as thenucleophile. PfOPRT and HsOPRT arecharacterized by late transition states withfully dissociated orotate, well-developed ribo-cations, and weakly bonded PPi nucleophiles(108). The N-ribosidic bond to the leavingorotate is 2.8 A, whereas weak participationof the PPi nucleophile gave a bond distanceof approximately 2.3 A (Figure 15). Thesephosphoribosyltransferase transition states aresimilar to but occur earlier in the reactioncoordinate than transition states for PfOPRTand HsOPRT previously determined with oro-tidine 5′-monophosphate and phosphonoaceticacid as substrates (109) and occur much later inthe reaction coordinate than a transition-statestructure reported for a bacterial OPRT usingorotidine 5′-monophosphate and phospho-noacetic acid (110). Similar transition statesfor PfOPRT and HsOPRT with different sub-strate analogs support similar transition-statestructures imposed by the enzymes. Geometricconstraints imposed by the catalytic sites mayimprint these transition states.

GENERAL APPLICABILITY OFENZYMATIC TRANSITION-STATE ANALYSIS

The examples provided above focus on N-ribosyltransferases. However, the method can

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I GG AC-GG-CC-GG-CC-G

A-14 (9-DA) 2'-OMe Ki* = 6 nMA-14 (9-DA) RNA Ki* = 4 nMA-14 (9-DA) DNA Ki* = 3 nM

N

N

HN

NH2

N

O

PO

O

O–

NH

N

NO

NH2N

O

OMeO

PO

O

HO

O–

N

NN

N

NH2

O

OH

NH

N

N

O

NH2N

O

OMeO

POO

O–

OMe

A GG AC-GG-CC-G

5' 3' 5' 3'

5' 3'

N

N

HN

NH2

N

OPOO

O–

NH

N

NO

NH2N

O

OMeOPOO

HO

O–

NH

N

NO

NH2N

O

OMeOPOO

O–

N

N

HN

NH2

NOPOO

O–

OPOO

O–

NH

N

NO

NH2N

O

OMeOPOO

O–

OH

A-10 RNA(substrate)

HN

N

NO

NO

O

O

P

OO

OH

N

OO

P OHO

HN

NN

O NH2

NH2

N

O

O

OP

O

HO

PO

HO

NN

N

N

O

O

O

OP

OHO

NO

R

R

R

N

N

HN

H2N

H2N

I GG AC-GG-CC-G

A-10 (9-DA) 2'-OMeKi* = 4 nM

Cyclic oxime G(9-DA)GA (R = 2'-OMe, DNA)Ki* = 4 nM

G(9-DA)GA 2'-OMeKi* = 9 nM

G(9-DA)Gs3 2'-OMeKi* = 7 nM

s3(9-DA)Gs3 2'-OMeKi* = 6 nM

N

N

HN

NH2

N

OH

HO

DADMeA (9-DA)Ki > 500 μM

HO HO

N

N

HN

NH2

N

OPOO

O–

OPOO

O–

OH

HO

s3(9-DA)s3Ki* = 690 nM

Figure 13Inhibitors for saporin L-1 and their dissociation constants. A-10 RNA is a substrate with 440 min−1 kcat and95 μM Km (reprinted from Reference 106 with permission). A-10, the 10-base RNA stem-loop shown in thefigure; DADMeA (9-DA), DADMe-immucillin-A, a transition state analog mimic for adenine-hydrolyzingribosome inactivating proteins when incorporated into RNA constructs; Ki

∗, the dissociation constant forinhibitors with saporin L-1; the asterisk indicates slow-onset inhibition is observed.


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ba

c d

Tyr80

Tyr123

Arg177

Tyr80

Tyr73

Tyr123

Glu177 Glu174

Tyr73

Arg134Arg134

3.53.1

5'

5'3'

3'

2.5

3.1

2.8

3.0

Water

WaterArg180

Figure 14(a) The inhibitor G(9DA)GA 2′-OMe is shown at the catalytic sites of ricin A chain and (b) saporin L-1.(c) In both cases, a catalytic site tyrosine [Tyr80 or (d ) Tyr73] is base stacked between the transition-stateanalog and the adjacent guanosine. The second tyrosine (Tyr123 in both cases) is base stacked on the otherside of the transition-state analog. The nucleophilic water is in contact with a catalytic site glutamic acid(Glu177 or Glu174) (reprinted from Reference 107 with permission).

be applied to any enzymatic reaction (see side-bar Beyond N-Ribosyltransferases).

TRANSITION-STATE VERSUSGROUND-STATE COMPLEXES

Glycosyltransferases are known to pass throughcationic transition states, and the resulting gly-cocations are often considered to be interme-diates with significant lifetimes (111). In somecases, experimental evidence exists for interme-diate status; for example, in the case of ricin, aribocationic intermediate is equilibrated withthe Michaelis complex (101, 102). A chemicalmodel for N-ribosyl hydrolysis has been pro-vided by the acid-catalyzed solvolysis of dAMPin 0.1 N HCl, where a deoxyribocation lifetimeof 10−11 to 10−10 s was estimated based on the8.5 to 1 ratio of α- to β-methyl-deoxyribose5-phosphate formed in the presence of 50%methanol as the nucleophile (112). The DNA

repair enzyme MutY, which hydrolyzes adeninefrom 8-oxo-G:A mismatches, is also reported toform a deoxyribocation intermediate with a life-time of less than 10−10 s (113). As human PNPis known to form a fully developed ribocation atthe transition state, computational studies havebeen employed to investigate the lifetime of theribocation and the accompanying reaction co-ordinate motion. As competitive, intrinsic KIEsare a two-state comparison (reactants free insolution are compared to the transition state),temporal and reaction coordinate informationis not provided, and computational chemistryapproaches are needed to provide additionalinsights.

Purine Nucleoside PhosphorylaseTransition-State Motion and Lifetime

Transition path sampling is an unbiased com-putational method used to locate transition

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states in complex systems (114, 115). Recently,it has been applied to the chemical steps oflactate dehydrogenase and human PNP (116).Starting conditions are provided by the crystalstructure of PNP with transition-state analogsat the catalytic sites. That this model is a reli-able estimate of the geometry near the transi-tion state is supported by the high success rateof unbiased barrier crossings using this method(17). Results of 220 barrier crossings show somecommon features associated with the transitionstate. Early loss of the ribosidic bond formsa ribocation transition state, followed by dy-namic local motion of the enzyme and the ri-bosyl group, causing migration of the anomericcarbon toward bound phosphate to form ri-bose 1-phosphate. Common features identi-fied by transition path sampling are (a) com-pression of the O4′. . .O5′ vibrational motionlinked to ribocation formation, (b) optimizedleaving group interactions, and (c) activationof the phosphate nucleophile as the reactionproceeds through the transition-state region.This complex reaction coordinate is completein approximately 70 femtoseconds (fs), withthe transition-state lifetime being only 10 fs(Figure 16). An important conclusion of thisstudy is that simultaneous optimization of theeffects needed to reach the transition state oc-cur on the femtosecond timescale and requirecoincident interactions for simultaneous ribo-cation formation and leaving group activation.

Test of the Reactive IntermediateHypothesis for Human PurineNucleoside Phosphorylase

The 10 fs transition-state lifetime (10−14 s) forhuman PNP is on the timescale of a single bondvibration and cannot be considered an inter-mediate. Because the diffusion of water is ap-proximately 1 A per picosecond (ps) (10−12 s),water would be unlikely to react with anenzyme-generated ribocation when phosphateis in the vicinity if the computational lifetimeof 10 fs is valid. If the lifetime of the ribo-cation is longer, for example, 10−10 s (100ps), water diffusion could easily capture the ri-

PfOPRT TS HsOPRT TS

2.8 Å2.8 Å

2.35 Å2.33 Å

Electronrich

Electrondeficient

Figure 15Transition-state structures of Plasmodium falciparum (PfOPRT) and human(HsOPRT) for the pyrophosphorolysis of orotidine. The reaction coordinatebond distances are shown above, and the electrostatic potential maps are shownbelow (red is electron rich, purple is electron deficient) (reprinted fromReference 108 with permission). TS, transition state.

bocation provided that solvent has access tothe catalytic site. Human PNP, like many en-zymes, has hydrophobic groups that cover thecatalytic site as a result of the loop motionassociated with substrate binding. One inter-pretation of burying substrates into enzymaticcatalytic sites is to shield reactive intermediatesfrom solvent. This hypothesis was tested forhuman PNP by systematically removing fouramino acid residues that sequester bound in-osine in the catalytic site from solvent water(117). Solvent leaks were introduced into the


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BEYOND N-RIBOSYLTRANSFERASES

The examples of enzymatic transition-state structures andtransition-state analogs discussed here are N-ribosyltransferases.Are KIE approaches to enzymatic transition-state structurelimited to certain classes of reactions? No, KIE analysis can beapplied to any chemical reaction, enzymatic or nonenzymatic,using approaches similar to those presented here. Are there ad-vantages to focusing on one class of reactions? Yes, a major effortin transition-state analysis is the synthesis of substrates with indi-vidual isotopic labels. An advantage of the N-ribosyltransferasesis the ability to use chemoenzymatic synthesis to convertlabeled glucose precursors into 5-phosphoribosyl-α-D-1-pyrophosphate and then to ATP. Additional enzymatic reactionsare available to convert the labeled ATP molecules into mostother purine and pyrimidine nucleosides and nucleotides. Otherglycosyltransferases are prime targets for transition-state analysisbecause many sugars can be converted from labeled glucose orare available with isotopic labels. Primary, α-secondary and β-secondary isotope effects give information on the extent of bondbreaking at the transition state, the degree of rehybridizationat the anomeric carbon, and the geometry of the sugar ring atthe transition state. Similar logic can be directly extended to allbiological reactions involving substitutions at carbon. New NMRand mass spectrometry techniques are being developed to permitaccurate analysis of natural abundance isotope effects. Thesemethods will expand the experimental access to transition-stateanalysis. Expansion of enzymatic transition-state knowledge willcontribute to the next generation of drug discovery efforts.

catalytic site by replacing individual catalyticsite–sequestering amino acids with glycine. Re-activity of the ribocation transition state wastested for capture by water relative to phosphatein the glycine mutants. NMR was used to fol-low ribose and ribose 1-phosphate formationfor approximately 106 catalytic cycles. Withoutphosphate, the phosphate-binding site fills withwater, and inosine is hydrolyzed by native PNPas well as by the “leaky” Y88G, F159G, H257G,and F200G enzymes. Hydrolysis did not occurin any of the leaky mutants when phosphate isbound despite the solvents’ access to the cat-alytic site. The result supports a ribocation life-time too short to permit water diffusion into thecatalytic site, a time estimated to be <5 ps (117).

An unprecedented N9-to-N3 isomerizationof inosine is catalyzed by H257G and F200Gin the presence of phosphate and by all PNPsin the absence of phosphate. Expansion of thecatalytic site permits ribocation formation withrelaxed geometric constraints, permitting nu-cleophilic rebound and N3-inosine isomeriza-tion (Figure 17).

Thermodynamics of Transition-StateAnalog Binding

Thermodynamic views of enzymatic transition-state theory suggest that the limiting affinityfor binding transition-state analogs is the sub-strate affinity enhanced by magnitude of theenzymatic rate enhancement (118, 119). ForPNP, the limiting dissociation constant is es-timated to be 6 × 10−18 M based on the rateof spontaneous cleavage of the glycosidic bondof adenosine (120), equivalent to a �G bindingof −23.6 kcal/mol. This value assumes a perfectmimic of the transition state, a physical impos-sibility because the transition state has nonequi-librium bond lengths and angles.

Transition-state analog binding to humanPNP has picomolar affinity, and the thermo-dynamics of binding to the first catalytic sitehave been analyzed by isocalorimetry (121).Inhibitor binding exhibits negative coopera-tivity, and inhibition of PNP catalytic activ-ity is complete when the inhibitor binds tothe first of the three subunits. Binding ofthe structurally rigid first-generation ImmH(Kd = 56 pM) is driven by negative enthalpyof �H = −21.2 kcal/mol, teasingly close tothe limit for a perfect transition-state analog,a �G binding of −23.6 kcal/mol. The �H of−21.2 kcal/mol is significantly greater than the�H of 18.6 kcal/mol or �G of 14.3 kcal/molmeasured for kcat during the chemical stepof PNP (122). However, an entropic (-T�S)penalty of 7.1 kcal/mol for ImmH binding pro-vides a �G binding of −14.1 kcal/mol, almostexactly matching the �G for kcat.

DADMe-ImmH (Kd = 9 pM) has increasedconformational flexibility compared to ImmH,and it binds with a reduced entropic penalty

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a Reactant b Step I: N-ribosidic bond cleavage

c Step II: ribosyl migration

e

d Product

His257

His257 His257

His257

Asn243

Asn243

Asn243 Asn243

Guanosine

Phosphate

Phosphate

Phosphate

Ribooxacarbenium ion

Ribose-1 phosphate

Ribooxacarbenium ion

Guanine

Guanine

Guanine

Glu201

Glu201

Glu201

Glu201

2.89

2.93

3.13

1.91

2.742.79

3.36

2.82

3.32

3.29 2.60

3.36

3.203.05

2.56

3.01

3.07

3.27

2.97

3.29 3.57

2.86

2.75

2.58

2.62

3.32

3.03

2.90

2.73

3.27

1.0

1.0

0.8

0.6

0.4

0.2

0

0.8

0.6

0.4

0.2

00 20 40 60 80 100 120 140 160

56 58 60 62 64 66 68 70

180 200 220 240

Time slice (fs)

Prob

abili

ty o

f pro

duct

form

atio

n

Figure 16(a-d ) Catalytic sitecontacts duringcatalysis in humanpurine nucleosidephosphorylase. (a) Thereaction involvescompression of threeoxygens from the 5′-O,4′-O and phosphategroups, (b) loss of theN-riboside bond,(c) ribosyl migration,and (d ) formation ofthe new C-O bond tophosphate. (e) Thereaction coordinatelifetime isapproximately 70 fs(50 fs shown here), andthe transition state (0.5probability of productformation) isapproximately 10 fs(reprinted fromReference 5 withpermission). Thelimits of zero (0) andunity (1.0) in(e) represent stablereactants and fullyformed products,respectively. The insetin (e) provides a moredetailed plot of theprobability of productformation at 55 to70 fs.


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Adenine

Phosphate

Ribocation

F200

F200

H257

E201N243

W2.58

8.44

4.45

2.82

3.13

3.49

2.93

2.84

3.24

2.90

3.35

2.67

W

H257

ImmHImmH

PO4

PO4

Y88

Y88

F159

F159

Human PNPtransition state

N9 return

N3 isomerization

HydrolysisPhosphorolysis

F200G

H257G

F159G

Y88G

PNP

Inosine

Ribose 1-P

I-13C NMR

F

E

D

C

B

A

98 97 96 95 94 93 92 91 90 89 88 87 ppm

N3-inosine Inosine

+

a

b

c

Figure 17Experimental test ofwater access to theribocation transitionstate of human purinenucleosidephosphorylase (PNP).The catalytic site ofhuman PNP is coveredby F200, H257, F159,and Y88. Replacementof each by glycineintroduces leaks intothe catalytic site (a).After approximatelyone million catalyticturnovers, 13C-NMR(b) demonstrates thatribose is not formed;thus, water cannotreact because thecarbocation lifetime istoo short to permitwater diffusion. Thesechemical reactions,including theunprecedentedformation ofN3-inosine, aresummarized in(c) (derived fromReference 117).

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16

12

8

4

–4

–8

–12

–16

–18

0

kcal

mol

–1Catalysis

human PNP

EA

E + I

EI‡

ΔGI‡ 15.1

ΔHI‡ –18.6

TΔSI‡ 3.5DADMe-ImmHbinding

EA‡

ΔG‡ 14.3

ΔH‡ 18.6

TΔS‡ –4.3

Figure 18Thermodynamics of transition-state barrier crossingcompared to transition-state analog binding forDADMe-ImmA and human purine nucleosidephosphorylase (PNP). The upper portion of the plot(red ) reports the thermodynamics for the chemicalstep (single-turnover kinetics), and the lowerportion (blue) reports the energetic signature oftransition-state analog binding to the first catalyticsite of human PNP-PO4. Numerical values are inkcal/mol. E, PNP; A, guanosine; �G, Gibbs freeenergy; �H, enthalpy; T�S, entropy;I, DADMe-ImmA.

of 4.3 kcal/mol but is dominated by the �H of−18.6 kcal/mol (Figure 18). The thermody-namic binding profile for DADMe-ImmH is anear perfect inverse reflection of the catalyticenergy barrier. Entropic penalties arise fromprotein and/or inhibitor conformational lossand changes in solvent organization, but suchpenalties are small compared to enthalpicenergy, emphasizing the large electrostaticcontributions to both catalysis and inhibitor

binding. H/D exchange and sedimentationvelocity confirmed that ImmH, with a largeentropy penalty, forms the least dynamic andstructurally tightest complex with humanPNP (123). DATMe-ImmH, a highly flexibleinhibitor, shows a smaller entropic penaltyof 2.3 kcal/mol, less than that for ImmHor DADMe-ImmH (123). The results haveimplications for catalysis by suggesting thatenzyme-inhibitor conformational flexibilitycan assist in tight binding and that tight bindingof transition-state analogs is compatible withdynamic protein flexibility. Computationalmolecular dynamics have provided a detailedlook at the conformational differences forPNP in substrate and different transition-stateanalog complexes (124). The favorable con-tribution of entropy to barrier crossing alsosuggests dynamic assistance in barrier crossing.

SUBSTRATE ANDTRANSITION-STATE BINDINGISOTOPE EFFECTS

The bond vibrational environment of indi-vidual atoms at enzymatic transition statesis probed by KIE experiments, whereasthose for binding interactions are probed byequilibrium BIEs (33, 34). By comparing

PNP + [5'–3H] ligand PNP• [5'–3H] ligand

N

N N

O

O

NH

OH

3H

OH

HO

NH

N

O

NH2+

NH

OH

3H

OH

HO

NH

N

O

NH+

NH3H

OH

HO

Inosine, Km = 40 µMBIE = 1.5%KIE = 4.7%

BIE = 12.6% BIE = 29.2%

ImmH, Kd = 58 pM DADMe-ImmH, Kd = 11 pM

Figure 19Binding isotope effects (BIEs) compared to the kinetic isotope effect (KIE) fortritium at the 5′-position of human purine nucleoside phosphorylase (PNP).The KIE is from bond changes between the Michaelis complex and thetransition state of purine nucleoside phosphorylase, and the BIEs are fromequilibrated thermodynamic complexes between unbound inosine (with boundSO4 as a phosphate mimic) and the Michaelis complex. For inhibitors, theequilibrium is between unbound and bound complexes with PNP-PO4(reprinted from Reference 125 with permission). DADMe-ImmH, DADMe-immucillin-H; ImmH, immucillin-H.


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KIE and BIE experiments, distortion of in-osine in the Michaelis complex can be com-pared to distortion of inosine at the tran-sition state of human PNP. Binding of[5′-3H]inosine to human PNP gives a BIEof 1.5%, but transition-state formation causesan intrinsic KIE (from transition-state distor-tions) of 4.7%. These values reflect atomicvibrational distortions in the 5′-C-H bondsat the Michaelis complex and the transitionstate. The degree of atomic distortion forcatalysis was compared to that for binding oftransition-state analogs. [5′-3H]immucillin-Hand [5′-3H]DADMe-immucillin-H gave large5′-3H BIEs of 12.6% and 29.2%, respectively(Figure 19). This unprecedented result re-vealed weak C5′-H distortion at the transitionstate and strong distortion in complexes withtransition-state analogs. A dynamic model ofcatalysis without the need for tight binding

at the transiton state is consistent with theseresults (125).

CONCLUSIONS ANDTHE FUTURE

Enzymatic transition states are accessible withthe combined use of intrinsic KIEs and compu-tational chemistry. Molecular electrostatic po-tential maps of reactants at enzymatic transi-tion states provide blueprints for the design oftransition-state analogs. Several inhibitors de-signed by these methods are in clinical trials.These early successes in drug design supportfuture contributions of transition-state analy-sis and inhibitor design to drug development.Access to transition-state analogs has also pro-vided structural access to difficult targets andinsights to thermodynamic contributions forbinding and catalysis.

SUMMARY POINTS

1. Closely related enzymes with near-identical catalytic sites can form distinct transitionstates.

2. Transition-state analysis from kinetic isotope effects (KIEs) and computational chemistrypermits the design of isozyme-specific transition-state analogs.

3. Inhibitors designed from the first principles of transition-state theory are among themost powerful noncovalent enzymatic inhibitors.

4. Transition-state analog inhibitors designed to bind to specific enzymatic targets arefinding applications in clinical trials.

5. Transition-state analogs can demonstrate binding enthalpies greater than the �G‡ bar-rier for catalysis.

6. The purine nucleoside phosphorylase transition state has a lifetime of 10 fs (10−14 s),whereas enzymatic complexes with transition-state analogs have lifetimes of 1 h (103 to104 s).

7. Binding of transition-state analogs causes remarkable distortion of analogs together withthe organization and stabilization of the enzymatic protein.

FUTURE ISSUES

1. How specific are transition-state analogs? Because analogs capture unique catalytic fea-tures of an enzyme, they may target either single or a few enzymes in the human catalyticrepertoire.

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2. Biological lifetimes of transition-state analogs are often longer than predicted by thedissociation rates of enzyme-inhibitor complexes. The mechanisms for analog recyclingare not well known, do not follow the normal rules of pharmacokinetics, and will requireparallel in vitro and in vivo studies.

3. Time constants for the range of protein dynamics span over 10 orders of magnitude.Which of these are most closely linked with transition-state formation, binding events,and interactions of transition-state analogs including slow-onset binding and inhibitordissociation?

4. Transition-state analogs commonly demonstrate slow-onset, tight-binding inhibition.Experimental approaches are needed to distinguish initial and final complex structures.

DISCLOSURE STATEMENT

The author is a consultant to pharmaceutical companies developing transition-state analog in-hibitors for clinical applications, an employee of Albert Einstein College of Medicine, and theholder of multiple patents based on transition-state analogs. Many of these patents have beenlicensed by the college to the pharmaceutical industry. The author receives royalty income fromthe development of these patents.

ACKNOWLEDGMENTS

V.L.S. is supported by National Institutes of Health research grants GM41916, CA072444,AI049512, and CA135405, as well as by National Institutes of Health program project P01-GM068036.

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for understanding enzyme catalysis? Proteins 78:1339–756. Hammes-Schiffer S, Benkovic SJ. 2006. Relating protein motion to catalysis. Annu. Rev. Biochem.

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BI80-FrontMatter ARI 16 May 2011 14:13

Annual Review ofBiochemistry

Volume 80, 2011Contents

Preface

Past, Present, and Future Triumphs of BiochemistryJoAnne Stubbe � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �v

Prefatory

From Serendipity to TherapyElizabeth F. Neufeld � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 1

Journey of a Molecular BiologistMasayasu Nomura � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �16

My Life with NatureJulius Adler � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �42

Membrane Vesicle Theme

Protein Folding and Modification in the MammalianEndoplasmic ReticulumIneke Braakman and Neil J. Bulleid � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �71

Mechanisms of Membrane Curvature SensingBruno Antonny � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 101

Biogenesis and Cargo Selectivity of AutophagosomesHilla Weidberg, Elena Shvets, and Zvulun Elazar � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 125

Membrane Protein Folding and Insertion Theme

Introduction to Theme “Membrane Protein Folding and Insertion”Gunnar von Heijne � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 157

Assembly of Bacterial Inner Membrane ProteinsRoss E. Dalbey, Peng Wang, and Andreas Kuhn � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 161

β-Barrel Membrane Protein Assembly by the Bam ComplexChristine L. Hagan, Thomas J. Silhavy, and Daniel Kahne � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 189

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Transmembrane Communication: General Principles and Lessonsfrom the Structure and Function of the M2 Proton Channel, K+

Channels, and Integrin ReceptorsGevorg Grigoryan, David T. Moore, and William F. DeGrado � � � � � � � � � � � � � � � � � � � � � � � � 211

Biological Mass Spectrometry Theme

Mass Spectrometry in the Postgenomic EraBrian T. Chait � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 239

Advances in the Mass Spectrometry of Membrane Proteins:From Individual Proteins to Intact ComplexesNelson P. Barrera and Carol V. Robinson � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 247

Quantitative, High-Resolution Proteomics for Data-DrivenSystems BiologyJurgen Cox and Matthias Mann � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 273

Applications of Mass Spectrometry to Lipids and MembranesRichard Harkewicz and Edward A. Dennis � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 301

Cellular Imaging Theme

Emerging In Vivo Analyses of Cell Function UsingFluorescence ImagingJennifer Lippincott-Schwartz � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 327

Biochemistry of Mobile Zinc and Nitric Oxide Revealedby Fluorescent SensorsMichael D. Pluth, Elisa Tomat, and Stephen J. Lippard � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 333

Development of Probes for Cellular Functions Using FluorescentProteins and Fluorescence Resonance Energy TransferAtsushi Miyawaki � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 357

Reporting from the Field: Genetically Encoded Fluorescent ReportersUncover Signaling Dynamics in Living Biological SystemsSohum Mehta and Jin Zhang � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 375

Recent Advances in Biochemistry

DNA Replicases from a Bacterial PerspectiveCharles S. McHenry � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 403

Genomic and Biochemical Insights into the Specificity of ETSTranscription FactorsPeter C. Hollenhorst, Lawrence P. McIntosh, and Barbara J. Graves � � � � � � � � � � � � � � � � � � � 437

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Signals and Combinatorial Functions of Histone ModificationsTamaki Suganuma and Jerry L. Workman � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 473

Assembly of Bacterial RibosomesZahra Shajani, Michael T. Sykes, and James R. Williamson � � � � � � � � � � � � � � � � � � � � � � � � � � � � 501

The Mechanism of Peptidyl Transfer Catalysis by the RibosomeEdward Ki Yun Leung, Nikolai Suslov, Nicole Tuttle, Raghuvir Sengupta,

and Joseph Anthony Piccirilli � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 527

Amyloid Structure: Conformational Diversity and ConsequencesBrandon H. Toyama and Jonathan S. Weissman � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 557

AAA+ Proteases: ATP-Fueled Machines of Protein DestructionRobert T. Sauer and Tania A. Baker � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 587

The Structure of the Nuclear Pore ComplexAndre Hoelz, Erik W. Debler, and Gunter Blobel � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 613

Benchmark Reaction Rates, the Stability of Biological Moleculesin Water, and the Evolution of Catalytic Power in EnzymesRichard Wolfenden � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 645

Biological Phosphoryl-Transfer Reactions: Understanding Mechanismand CatalysisJonathan K. Lassila, Jesse G. Zalatan, and Daniel Herschlag � � � � � � � � � � � � � � � � � � � � � � � � � � � 669

Enzymatic Transition States, Transition-State Analogs, Dynamics,Thermodynamics, and LifetimesVern L. Schramm � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 703

Class I Ribonucleotide Reductases: Metallocofactor Assemblyand Repair In Vitro and In VivoJoseph A. Cotruvo Jr. and JoAnne Stubbe � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 733

The Evolution of Protein Kinase Inhibitors from Antagoniststo Agonists of Cellular SignalingArvin C. Dar and Kevan M. Shokat � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 769

Glycan Microarrays for Decoding the GlycomeCory D. Rillahan and James C. Paulson � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 797

Cross Talk Between O-GlcNAcylation and Phosphorylation:Roles in Signaling, Transcription, and Chronic DiseaseGerald W. Hart, Chad Slawson, Genaro Ramirez-Correa, and Olof Lagerlof � � � � � � � � � 825

Regulation of Phospholipid Synthesis in the YeastSaccharomyces cerevisiaeGeorge M. Carman and Gil-Soo Han � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 859

Contents ix

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Sterol Regulation of Metabolism, Homeostasis, and DevelopmentJoshua Wollam and Adam Antebi � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 885

Structural Biology of the Toll-Like Receptor FamilyJin Young Kang and Jie-Oh Lee � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 917

Structure-Function Relationships of the G Domain, a CanonicalSwitch MotifAlfred Wittinghofer and Ingrid R. Vetter � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 943

STIM Proteins and the Endoplasmic Reticulum-PlasmaMembrane JunctionsSilvia Carrasco and Tobias Meyer � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 973

Amino Acid Signaling in TOR ActivationJoungmok Kim and Kun-Liang Guan � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �1001

Mitochondrial tRNA Import and Its Consequencesfor Mitochondrial TranslationAndre Schneider � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �1033

Caspase Substrates and Cellular RemodelingEmily D. Crawford and James A. Wells � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �1055

Regulation of HSF1 Function in the Heat Stress Response:Implications in Aging and DiseaseJulius Anckar and Lea Sistonen � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �1089

Indexes

Cumulative Index of Contributing Authors, Volumes 76–80 � � � � � � � � � � � � � � � � � � � � � � � � � �1117

Cumulative Index of Chapter Titles, Volumes 76–80 � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �1121

Errata

An online log of corrections to Annual Review of Biochemistry articles may be found athttp://biochem.annualreviews.org/errata.shtml

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