the mechanism of peptidyl transfer catalysis by the ribosome

BI80CH23-Piccirilli ARI 16 May 2011 12:23

The Mechanism of PeptidylTransfer Catalysis bythe RibosomeEdward Ki Yun Leung,1 Nikolai Suslov,1

Nicole Tuttle,2 Raghuvir Sengupta,3

and Joseph Anthony Piccirilli1,2

1Department of Biochemistry and Molecular Biology, 2Department of Chemistry,The University of Chicago, Chicago, Illinois 60637; email: [email protected] of Biochemistry, Stanford University, Stanford, California 94305

Annu. Rev. Biochem. 2011. 80:527–55

First published online as a Review in Advance onMay 6, 2011

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

This article’s doi:10.1146/annurev-biochem-082108-165150

Copyright c© 2011 by Annual Reviews.All rights reserved

0066-4154/11/0707-0527$20.00

Keywords

translation, peptide bond formation, induced fit, peptidyl transferasecenter, entropy trap

Abstract

The ribosome catalyzes two fundamental biological reactions: peptidyltransfer, the formation of a peptide bond during protein synthesis, andpeptidyl hydrolysis, the release of the complete protein from the pep-tidyl tRNA upon completion of translation. The ribosome is able toutilize and distinguish the two different nucleophiles for each reac-tion, the α-amine of the incoming aminoacyl tRNA versus the watermolecule. The correct binding of substrates induces structural rear-rangements of ribosomal active-site residues and the substrates them-selves, resulting in an orientation suitable for catalysis. In addition,active-site residues appear to provide further assistance by orderingactive-site water molecules and providing an electrostatic environmentvia a hydrogen network that stabilizes the reaction intermediates andpossibly shuttles protons. Major questions remain concerning the tim-ing, components, and mechanism of the proton transfer steps. Thisreview summarizes the recent progress in structural, biochemical, andcomputational advances and presents the current mechanistic modelsfor these two reactions.

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Contents

1. INTRODUCTION . . . . . . . . . . . . . . . . 5281.1. Overview of Translation . . . . . . . 529

2. THE PEPTIDYL-TRANSFERREACTION CATALYZED BYTHE RIBOSOME. . . . . . . . . . . . . . . . . 529

3. NONENZYMATIC PEPTIDEBOND FORMATION . . . . . . . . . . . . 531

4. THE pH DEPENDENCY OFTHE RIBOSOME-CATALYZEDREACTION . . . . . . . . . . . . . . . . . . . . . . 534

5. INDUCED-FIT ANDSUBSTRATE-ASSISTEDCATALYSIS . . . . . . . . . . . . . . . . . . . . . . 537

6. ENTROPIC AND ENTHALPICACTIVATION OF THEPEPTIDYL TRANSFERREACTION . . . . . . . . . . . . . . . . . . . . . . 542

7. PEPTIDE RELEASE . . . . . . . . . . . . . . 5457.1. Catalysis of Peptide Release . . . . 545

8. CONCLUSION ANDOUTLOOK . . . . . . . . . . . . . . . . . . . . . . 547

1. INTRODUCTION

The central dogma of biology posits that infor-mation flows from DNA to RNA to protein.The ribosome serves as the exquisitely complexmolecular machine that decodes RNA and usesthe information to build a polypeptide contain-ing a precise sequence of amino acids. Ribo-somes play a central role in all living organisms,and their discovery earned George Emil Palade,Albert Claude, and Christian de Duve the 1974Nobel Prize in Physiology or Medicine. Abroad range of perspectives has fueled scientificinquiry into ribosome structure and functionover the years, from identifying causes of beecolony destruction (1) to designing futuristicnanomachines (2). As an enzyme containing anactive site composed of RNA, ribosomes holdspecial significance from an evolutionary view-point as molecular fossils of the so-called RNAWorld era when organisms relied on RNAfor information storage and catalytic func-

tion. Gradually, protein enzymes supplantedribozymes, presumably via the formation of ri-bonucleoprotein particles (RNPs). Ribosomestherefore may represent a relic of the transitionfrom the RNA World to the RNP World.From the perspective of human health, the ri-bosome serves as an attractive medicinal targetfor drugs that work by inhibiting translationwithin pathogens without affecting the trans-lational apparatus of the host organism. Withrespect to the academic quest of understandingthe molecular foundation of living systems,unraveling of the molecular underpinnings ofribosome structure and function represents atriumph and a paradigm for modern molecularbiology. In 2009, Venkatraman Ramakrishnan,Thomas A. Steitz, and Ada E. Yonath wereawarded the Nobel Prize in Chemistry fortheir work on the structure and function of theribosome. The atomic-resolution structure ofthe ribosome once seemed out of reach, but asof today, we not only have views of the pep-tidyltransferase center (PTC), but we also havesnapshots of the ribosome at different stages ofpolypeptide assembly. These structural insightsculminate in the ability to build an action movieof the translation process, which, althoughfictional in some respects, illuminates theimmense power of structural biology to impartphysical meaning to molecular processes.

As large RNPs consisting of three ribosomalRNA (rRNA) molecules and more than 50 pro-teins, ribosomes rapidly and accurately selectaminoacyl tRNAs and catalyze the synthesis ofpolypeptides from amino acids. Prokaryotic ri-bosomes consist of two subunits, the large 50Ssubunit and the smaller 30S subunit; togetherthey form the 2.5 mDa 70S ribosome. The 50Ssubunit contains the 5S (∼120 nucleotides) and23S (∼2,900 nucleotides) rRNA molecules andmore than 30 proteins; the 30S subunit consistsof the 16S (∼1,500 nucleotides) and ∼20 pro-teins. Several other protein factors interact withthe ribosome at various stages of translation.This review features mainly the prokaryotic ri-bosome because much of our current mechanis-tic and structural understanding has developedfrom studies of bacterial and archeal ribosomes.

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1.1. Overview of Translation

The biological process of translation can be di-vided into three major stages: initiation, elonga-tion, and termination. The ribosome has threetRNA binding sites: A, P, and E sites (Figure 1).The 30S subunit mediates selection of cognateaminoacyl tRNAs by facilitating base-pairingbetween mRNA codons and tRNA anticodonswhile the active site, or the PTC, resides in the50S subunit. The PTC catalyzes both peptidyltransfer during protein elongation and hydrol-ysis of the peptidyl tRNA during termination.

Initiation requires the formation of the ini-tiation complex, where base-pairing of the 30S16S rRNA 3′ end with the nearly complemen-tary Shine-Dalgarno sequence just upstreamof the start codon positions the start codon inthe P site for the binding of fMet-tRNAfMet,and three initiation factors (IFs), 1, 2, and 3.Subsequent joining of the 50S subunit, GTPhydrolysis by IF2, and dissociation of IF1, -2,and -3 prime the ribosome for the elongationstage of translation. Elongation begins withthe aminoacyl tRNA delivered to the A site asa ternary complex with elongation factor Tu(EF-Tu) and GTP (Figure 1a). The correctbinding of the cognate aminoacyl tRNAtriggers a series of conformational changes inthe aminoacyl tRNA and EF-Tu and GTP hy-drolysis. EF-Tu dissociates from the ribosome,the “relaxed” aminoacyl tRNA subsequentlyundergoes accommodation, the proper posi-tioning of the aminoacyl 3′ end of the A-sitetRNA moves into the PTC, and peptide bondformation subsequently occurs. In the finalphase of elongation, the ribosomal E site, whichpreferentially binds deacylated tRNA and notpeptidyl tRNA (3–5), and GTP hydrolysismediated by another GTP-bound elongationfactor (EF-G•GTP) facilitate translocation.The deacylated P-site tRNA and the A-sitepeptidyl tRNA spontaneously translocatewhile maintaining their codon interactionsalong with other structural rearrangementsvia E/P and A/P hybrid states; this triggersa reversible counterclockwise ratchet-likerotation between the two subunits. Subsequent

EF-G•GTP binding to the ratcheted state andGTP hydrolysis reset the ratcheted ribosome.It is generally accepted that GTP hydrolysisprecedes translocation (6), but how GTP hy-drolysis leads to all these processes still remainsunclear. Presumably, GTP hydrolysis inducesa series of conformational rearrangements thatallow the movement of mRNA and tRNAs(7, 8). Elongation continues until a stop codonenters the A site, triggering the final stageof translation, termination. Release factors(RFs) recognize and bind the A-site stop codonand trigger the hydrolysis and release of thepolypeptide from the P-site tRNA. Subsequentbinding of EF-G•GTP and the ribosomerecycling factor leads to GTP hydrolysis anddissociation of the ribosomal subunits.

2. THE PEPTIDYL-TRANSFERREACTION CATALYZEDBY THE RIBOSOME

Enzymes commonly contain active-siteresidues that participate in the chemicaltransformation of their substrates. Functionalroles for such residues may include directparticipation in chemical catalysis, which mayinclude facilitation of proton transfer reactionsand covalent chemistry at the reaction center.Active-site residues may engage directly inthe formation of covalent bonds as generalacids, general bases, or nucleophilic catalysts.Additional functional roles may include elec-trostatic and structural complementarity tothe transition state (TS), desolvation or thereorganization of water molecules, and theuse of binding energy to lower the entropyof activation (9, 10). With the exception ofnucleophilic catalysis, which the ribosomedoes not use (11), any of these other catalyticstrategies may be employed.

During elongation, the ribosome PTCcatalyzes the aminolysis of an ester bond,where the α-amino group of A-site aminoacyltRNA nucleophilically attacks the P-sitepeptidyl tRNA at the carbonyl carbon of theester bond that links the peptide to the tRNA.Amines react intrinsically fast with esters to

www.annualreviews.org • Peptidyl Transfer Catalysis by the Ribosome 529

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EF-Tu

APE5' 3'

Binding of RF1/2

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CC O–

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dCdC

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c d

mRNA

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Small subunit

Figure 1Overview of steps leading to (a) peptidyl transfer and (b) peptide release, and (c,d ) structures of transition state analogs used to study theribosome. (a) Aminoacyl tRNA binds to the A site of the ribosome in the form of a “ternary complex” with elongation factor Tu(EF-Tu) and GTP. A recent crystal structure of EF-Tu and aminoacyl tRNA bound to the A site of the ribosome revealed thatdistortion of the aminoacyl tRNA enables its interactions with EF-Tu and the decoding center of the 30S subunit (121). The correctbinding of the cognate aminoacyl tRNA triggers a series of conformational changes in the aminoacyl tRNA and EF-Tu, suggesting apossible communication pathway between the decoding center and the GTPase center of EF-Tu. GTP hydrolysis by EF-Tu releasesthe aminoacyl tRNA from the EF-Tu-GDP. Prior to peptide bond formation, the aminoacyl tRNA undergoes an accommodation step,whereby its 3′-CCA end engages in multiple interactions with the large ribosomal subunit. Peptide bond formation subsequently occursand yields a deacylated tRNA in the P site and a peptidyl tRNA in the A site extended by one amino acid residue. (b) Release factor(RF)1 and RF2 recognize and bind to the stop codon on the mRNA A site. Binding of RF1/2 leads to hydrolysis and release of thepeptide from the tRNA in the P site. Structures of the Yarus (c) and RAP (d ) transition state analogs used in structural studies with the50S subunit of the Haloarcula marismortui ribosome by Nissen et al. (32) and Schmeing et al. (55). The puromycin moiety of the Yarustransition state analog is in red. Figure 1a,b adapted from Steitz (122), Figure 1c adapted from Nissen et al. (32), and Figure 1dadapted from Schmeing et al. (55).

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form peptide bonds (∼10−4 M−1s−1 at roomtemperature). The ribosome accelerates thisreaction by ∼106- to 107-fold (12). Beforepeptide bond formation, the aminoacyl tRNAbinds to the A site and accommodates at arate of ∼10 s−1 (13), significantly slower thanthe rate at which the ternary complex reactsto form a peptide bond (≥300 s−1) (14). Slowaccommodation confounds mechanistic studiesusing full-length aminoacyl-tRNA substrates,and this has led to the use of minimal substrateanalogs [short RNA fragments that mimicthe universally conserved tRNA CCA 3′ endand/or have the α-amine substituted with aweaker hydroxyl (OH) nucleophile]. Amongthese minimal substrates are puromycin (Pmn),C-puromycin (C-Pmn), and CC-puromycin(CC-Pmn); the additional cytidine residuescorrespond to C74 and C75 of tRNA. Theybind rapidly to the ribosome and react with theP-site bound substrate at rates up to 50 s−1 (15,16), thereby circumventing the limitations ofaccommodation and enabling direct analysis oftheir chemistry. However, as these analogs lackthe tRNA body, details of substrate positioningmay not fully mimic those for full-length tRNA.

Owing to recent progress in ribosomecrystallography and concurrent analysis usingkinetic, biochemical, genetic, and computa-tional approaches, a fairly consistent picture ofthe catalytic mechanism has begun to emerge.Nevertheless, significant questions remainconcerning the proton transfer steps. Whereasenzymatic reactions that involve general acidbase catalysis typically occur with a decreasedactivation enthalpy relative to the uncatalyzedreaction, the aminolysis reaction in the ribo-some ternary complex occurs with a greaterenthalpy of activation relative to the reaction insolution. Instead, catalysis of peptidyl transferby the ribosome accompanies a decrease in theentropy of activation relative to the uncatalyzedreaction (12). Therefore, a significant aspectof understanding ribosome catalysis involvesdefining the molecular mechanisms that renderthe activation entropy favorable, such assubstrate and water molecule positioning inthe active site, desolvation, and electrostatic

shielding (12). This review describes thecurrent understanding of these molecularmechanisms as they relate to peptidyl transferand peptide release in the PTC and thefundamental features of the correspondinguncatalyzed reactions.

3. NONENZYMATIC PEPTIDEBOND FORMATION

The study of the ribosome mechanism hasbenefited from the mechanistic frameworkdeveloped over decades from studies of amidebond formation in solution. A well-studiedmodel reaction involves the hydrazinolysisof methyl formate. In the proposed reactionmechanism (Figure 2a), hydrazine attacks thecarbonyl carbon nucleophilically to form azwitterionic tetrahedral intermediate (TI± ).Following deprotonation, the resulting anionicintermediate (T−) collapses with expulsion ofmethoxy anion. Hydroxide and other bases,including a second molecule of amine, can actas general bases to catalyze the deprotonationof T± (17). As summarized below, data fromkinetics, linear free-energy relationships (17,18), isotope effects (19–21), and computationalanalysis (22–25) support this mechanism.

Reactions of esters with aliphatic aminesgenerally show breaks in their pH-rate pro-files at pH 6.3–8.7, indicating a change in rate-limiting step and therefore the presence of anintermediate. For the reaction of hydrazine(pKa = 8.3) with methyl formate, this break oc-curs at pH 8.7 (17, 18). Below pH 8.7, the k1 andk3 steps are rapid and reversible. Breakdown ofT− has the highest energy TS relative to theground state and is thus rate-limiting. AbovepH 8.7, deprotonation of the zwitterionic T±

is rate-limiting; owing to the low concentrationof a suitable acid at high pH, it is more energet-ically favorable for T− to expel methoxy anion(k5) than to accept a proton and subsequentlyexpel the amine (Figure 2b).

Analysis of the reaction, using linear free-energy relationship approaches, has yieldedBrønsted coefficients (β) for the nucleophileand the leaving group under conditions


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H OCH3

O

H2NNH2

H2NN OCH3

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1.003, 1.004

1.0621, 1.0048

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klight/kheavy at pH 8 or pH 10

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Ester +amine

Amide

pH 8

Ester +amine

Amide

En

erg

y

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erg

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+

Figure 2Mechanism for the hydrazinolysis of methyl formate in water. (a) The reaction proceeds in three steps withtwo tetrahedral intermediates. This reaction proceeds with general base catalysis by a second molecule ofamine, represented here as B. (b) The energy diagrams of the reactions of esters with aliphatic amines at pH10 and 8 indicate a change in rate-limiting step and therefore the presence of an intermediate. (c) Isotopeeffect measurements are shown for five atoms in the reaction measured at pH 8 (red ) or pH 10 (blue).Figure adapted from Marlier et al. (26).

whereby formation of T− limits the reactionrate. β indicates how the basicity of a particulargroup influences the rate of a reaction and re-flects the change in effective charge on an atomas the reaction proceeds from the ground stateto the rate-limiting TS. General base-catalyzedaminolysis of alkyl esters by amines, such as hy-drazine, react with βnuc = 0.9 (17). The changein charge includes both the positive charge de-veloped by the amine nucleophile (k1) and sub-sequent transfer of that charge to a secondamine nitrogen during deprotonation (k3). Forthe effect of leaving-group pKa on the reactionrate, these same reaction steps give βLG = −0.6(18), reflecting the loss in resonance betweenthe ester oxygen and the carbonyl group uponT− formation.

Isotope effect data confirm the pH-inducedchange in the rate-limiting step and further sup-port the proposed mechanistic steps. Althoughisotope effects must be interpreted cautiously,they provide a highly sensitive measurement ofbonding changes to particular atoms in a reac-tion. In general, atoms that undergo a changeto a less stiff bonding environment (such as loss

of a leaving group) give isotope effects largerthan 1 and are referred to as “normal,” whereasatoms that experience an increase in bonding(such as attacking nucleophiles) generally giveisotope effects smaller than 1 and are referredto as “inverse.” Early work characterized thebonding changes for the methoxyl oxygen (20)and the formyl hydrogen (19); Marlier et al. (26)completed the picture by measuring isotope ef-fects at two different pH values for five differentatoms.

At pH 8, the leaving-group atom gives alarge, normal isotope effect of 1.0621, indi-cating significant C-O bond cleavage in thehighest energy TS, as expected for rate-limitingcollapse of T−. This isotope effect decreasessignificantly upon shifting the reaction pHto 10, where deprotonation of T± limits thereaction rate, indicating that C-O bond cleav-age must occur after the rate-limiting step. AtpH 10, the formyl hydrogen gives an inversesecondary isotope effect of 0.72, reflectingthe shift from sp2 to sp3 hybridization at thecarbonyl carbon as the reaction progressesfrom the ground state to the rate-limiting

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TS; at pH 8, this effect becomes significantlyless inverse, suggesting that T− collapses viaa late TS with the carbonyl carbon regainingsignificant sp2 character.

At pH 8, the carbonyl carbon gives a normalisotope effect owing to the loss of leaving group,as expected when breakdown of T− is ratelimiting. Because of the expectation that thecarbonyl carbon experiences a stiffer bondingenvironment as its hybridization changes fromsp2 in the ground state toward sp3 in the TS, aninverse effect was anticipated for formation ofT± or T−. Thus, the observed normal isotopeeffect at pH 10 was viewed initially as evidenceagainst Jencks’ stepwise mechanism (21).However, subsequent computational worksuggested that a normal isotope effect couldarise in this reaction owing to weakening andlengthening of the C-N+ bond in T± causedby hyperconjugative overlap of nonbondingelectrons of the ester oxygen with the C-N+

antibonding orbital (23). The nitrogen nu-cleophile has an inverse isotope effect at bothpH measurements, but the interpretation iscomplicated because either hydrazine nitrogencould act as the nucleophile and as a generalbase. Marlier (27) interpreted the observedinverse isotope effect as the result of a largeinverse equilibrium effect associated with for-mation of T± that is offset partially by a normaleffect from subsequent deprotonation of T± .

Although questions remain, the cumulativedata provide robust evidence for the mecha-nism outlined in Figure 2a. On the basis ofrecent isotope effect data, ribosomal substrateslikely react in solution through the same mech-anism. For the hydroxylaminolysis of CCA-phenylalanyl-caproic acid-biotin (CCApcb) atpH 8.5, Hiller et al. (28) measured isotopeeffects for the 2′-oxygen, 3′-oxygen leavinggroup, carbonyl oxygen and carbon, and α-hydrogen. The reported values follow the sametrends as the values measured by Marlier et al.(21) for hyrdrazinolysis of methyl formate. Thecarbonyl oxygen and the α-hydrogen yieldedisotope effects of 1.037 and 0.962, consis-tent with a tetrahedral TS as in Figure 2a.These data, combined with a 3′-oxygen

leaving-group isotope effect of 1.029, stronglysupport breakdown of T− as the rate-limitingTS. Of course, the solvent organization aroundthe reactants, TSs, and intermediates, as wellas the mechanism of solvent reorganizationalong the reaction trajectory, remains largelyunknown.

Intriguingly, the available physical organicdata for the ribosomal peptidyltransferase re-action differ from that measured for aminolysisin solution. Seila et al. (29) measured the 15Nkinetic isotope effect for the α-amine in theribosomal peptidyltransferase reaction, observ-ing normal isotope effects of 1.0090 and 1.0097at pH 5.2 and 8.5, in contrast to the inverse ef-fect of 0.99 for the solution aminolysis reaction(Figure 2a). Seila et al. (29) concluded the ob-served ribosomal nitrogen isotope effect is mostconsistent with a model in which nucleophilicattack and deprotonation of the amine occurconcurrently. An alternative model wherenucleophilic attack and deprotonation occurin consecutive steps, with the latter occuringbefore or during the rate-limiting step, alsoremains viable. In the future, these data will sup-port more definitive mechanistic conclusionsin the context of isotope effects for other atomsinvolved in the peptidyltransferase reaction.

Okuda and colleagues (30, 31) completeda Brønsted analysis of the peptidyltransferasereaction using Pmn derivatives with varyingα-amine pKas. With the 50S subunit, theyobtained a βnuc of 0.06 and values of −0.27 and0.06 with the 70S subunit in the absence andpresence of DMSO, respectively, suggestingthe α-amine bears little charge in the rate-limiting TS, consistent with implications fromthe 15N kinetic isotope effect. This evidencecould support a rate-limiting T−-like TS forthe ribosome reaction, whereby nitrogen hasthe same charge in the ground state and TS,or a mechanism whereby deprotonation of T±

and loss of the leaving group occur at the sametime. The ribosome measurement appears tocontrast the aminolysis of alkyl esters in solu-tion, which, as outlined above, occurs with βnuc

values of 0.7–0.9 for primary and secondaryamines (17). However, the βnuc values for the


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solution reaction include proton transfer toa second amine during general base catalysis,confounding direct comparison to the ribo-some reaction, whereby a second amino groupfrom another amino-acyl tRNA does not act asa general base.

In summary, peptide bond formation in so-lution follows the classic mechanism for acyltransfer and appears to require amine deproto-nation prior to departure of the leaving group.Ribosome-catalyzed peptide bond formationmost likely follows this general pathway, butthe timing and mechanism of proton trans-fer steps remain uncertain (Figure 3). Protontransfer from the amine could involve a generalbase (Figure 3b,c) or a proton shuttle network(Figure 3d,e) and occur concurrently withnucleophilic attack (Figure 3c,e) or follow-ing nucleophilic attack (Figure 3b,d ) eitherpreceding (Figure 3b) or concomitant withleaving-group expulsion (Figure 3d ). Manyvariations of these mechanistic themes are pos-sible, including pathways involving the 2′-OHgroup, one or more solvent molecules, T± ,T− and neutral intermediates, six- or eight-membered ring proton shuttle networks, andcombinations thereof, for nucleophilic attackand leaving-group departure steps in the mech-anism. Current data cannot rule out many ofthese possibilities. In addition, we have no in-formation about when protonation of the leav-ing group occurs or whether the carbonyl oxy-gen undergoes transient protonation along thereaction trajectory (Figure 3e). Although phys-ical organic data for the ribosomal reaction

deviate from the solution reaction, this likelyreflects differences in the rate-limiting step andthe timing and pathway of the proton trans-fer steps rather than a fundamental differencein the reaction mechanism. In addition to thetiming issue, identifying the active-site compo-nents that mediate amine deprotonation andother proton transfer steps remains an activeand challenging area of research, as describedin the next section.

4. THE pH DEPENDENCY OFTHE RIBOSOME-CATALYZEDREACTION

The first atomic-resolution structure of theHaloarcula marismortui 50S subunit [bound witha transition state analog (TSA), the Yarus com-pound, Figure 1c] led Moore and colleagues(32) to hypothesize that the ribosome utilizes ageneral acid-base catalytic mechanism. In thestructure, the N3 atom of A2451 (Escherichia colinumbering), a universally conserved residue,appears to make direct contact with the nascentpeptide bond (Figure 4a). In conjunctionwith this observation, dimethylsulfate (DMS)mapping data of E. coli ribosomes, whichsuggested that the pKa of A2451 approachesneutrality, inspired the proposal that A2451 N3acts as a general acid-base catalyst, whereby itdeprotonates the A-site nucleophilic α-amineand, subsequently, serves as a general acidby donating a proton to the P-site 3′-oxygenleaving group (32, 33). As most efficient generalacid-base catalysts usually have pKas close to

−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−→Figure 3A subset of possible mechanisms for catalysis of peptide bond formation in the peptidyltransferase center. (a) Two-intermediatemechanism analogous to the solution reaction with no explicit proton transfer steps shown. (b) Two-intermediate mechanism withgeneral base and general acid catalysis. The general base (B) facilitates proton transfer from the attacking amine in the T± . Thegeneral acid (BH+) facilitates proton transfer either to the leaving group as shown or to the oxyanion (not shown). A single entity maymediate both proton transfers, or B and BH+ may represent distinct entities. Additionally, B or BH+ may mediate proton transferdirectly as shown or indirectly via water molecules or active-site functional groups (for example, see shuttling mechanisms in d and e).(c) One-intermediate mechanism with general base and general acid catalysis. Nucleophilic attack and proton transfer to the generalbase occur in the same step, bypassing formation of T± . (d ) One-intermediate mechanism with 2′-OH-mediated proton transfer fromthe T− amino group to the leaving group via a six-membered ring shuttle network; and (e) proton shuttle mechanism involving aneutral amide hemiacetal (T0) intermediate. Distinct water molecules mediate T0 formation and breakdown via six-membered ringproton shuttle networks. Colors are used to show the transfer of individual hydrogen atoms.

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neutrality under physiological conditions (10),Moore and colleagues (32) proposed that acharge-relay network, involving G2447 andthe A2450 phosphate group, shifts the pKa ofthe acidic N3 atom of A2451 (pKa ≈ 1) towardneutrality.

Although these initial observations providedevidence that A2451 could act as a generalacid base, subsequent observations called thismodel into question. First, ribosomes fromother organisms do not show the observed pH-dependent A2451 DMS reactivity, and the pH

a

b

c

d

e

O

OHOO

N HH

R

O

O

O

Ade AdeAde

Ade Ade

O NR

H

H

T

H

NHO

O

OHHO

R

O

OHO

Ade

O

T

NH

R

O

OH

Ade

R

NH

O

B H B

N

H

H

R

B

O

OHO

Ade

O

O

OHO

Ade

OR

O

OHO

Ade

ON

H

H

R

T

O

OHO

Ade

O

T

NH

R

O

OHO

Ade

R

NH

O

O

OH

Ade

R

NH

O

O

OHO

Ade

O

H2N

H2N

R

O

OHO

Ade

ON

H

H

R

T

O

OHO

Ade

O

T

NH

R

O

OHHO

HO

HO

Ade

R

NH

OB

B H B

B

O

OHOO

N R NHOH

H

H

OH

O

OHOO

NH

RH

O H

H

O H

H

OH

H

O

OHHO

Ade

O

H

OH

H

HR

T0


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a b

U2620(2585)

G2618(2583)

methylU2619(2584)

U2620(2585)

U2541(2506)

pep

Phe A76

C75

P site

Approachof attack

U2541(2506)

A2486(2451)

Pmn

A2637(2602)

3 Å

Preinduced position

Bound position

Transition state analog

Figure 4Crystal structure of H. marismortui peptidyltransferase active site. (a) A view of puromycin (Pmn) bound to the A site surrounded by23S rRNA residues A2486, U2541, U2620, and A2637 (Protein Data Bank 1FG0; E. coli numbering and not all active-site residues areshown). The N3 of A2486 of this structure is ∼3 A away from the carbonyl center, leading to the initial hypothesis that the ribosomeutilizes general acid-base catalysis. (b) The conformation change induced in G2618 by A-site substrate binding breaks its G-U wobblepair with U2451, which swings through 90◦. methylU2619 and U2620 shift, allowing the ester group to move from its preinducedposition within the CCA-phenylalanyl-caproic acid-biotin (CCApcb) (light green) Ch-Pmn ternary complex to that observed within theCCApcb (medium green) CCh-Pmn ternary complex. Following attack by the A-site substrate, the ester group presumably adopts theposition observed for the bound transition state analog (black). The growing peptide chain (pep) does not appear to undergo anysignificant conformational change. Reprinted with permission from Macmillan Publishers Ltd., Nature (49), copyright 2005.

dependency of DMS reactivity varies with tem-perature and monovalent ion concentration,suggesting that pH-dependent conformationalchanges within the PTC govern A2451 reac-tivity (33, 34–37). Second, if A2451 played acritical role during peptidyl transfer, mutationof this residue would be expected to have a dele-terious effect. Although base substitutions atA2451 conferred a dominant lethal phenotypewhen expressed, A2451 mutants accumulatedin polysomes (38, 39) and retained significantenzymatic activity with full-length tRNAs invitro (40). Furthermore, using an in vitro re-constitution system that allows incorporationof nonnatural nucleosides into the 23S rRNA,Erlacher et al. (41) showed that removal of theN3 atom and the nucleobase of A2451 hadminimal effects on peptide bond formation.

Consistent with these observations, mutationof G2447, a residue implicated in the charge-relay network, had no effect on cell viabilityand had modest effects on enzymatic activity(38, 42).

Although the biochemical data describedabove provide evidence against A2451 nucle-obase catalysis, they do not rule out the possibil-ity that other ribosome residues provide generalacid-base catalysis. The pH dependency of a re-action relates to the number of protonation ordeprotonation events prior to the rate-limitingstep and, in certain circumstances, may revealpKa values of ionizing groups in enzymes (43).To extract meaningful conclusions from pHtitrations, experiments must monitor the sameelementary step or group of steps over the entirepH range and test the multiple mechanisms that

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could account for the observed pH dependency(43–45). These concerns pertain especiallyto the ribosome, a complex macromolecularmachine known to undergo pH-dependentconformational changes (33, 34–37).

Presteady-state kinetics for the reaction be-tween a P-site dipeptidyl tRNA and A-site Pmnindicates that the rates depend on the pH.Katunin et al. (16) observed that the single-turnover rate constant for the catalytic step,kpep, has a steep dependency on pH (slope ∼2below pH 7.5) with two ionizable groups (pKa1

of 6.9 and pKa2 of 7.5). pKa1 likely reflects thetitration of the nucleophilic amine of Pmn be-cause (a) it matches the solution pKa of Pmn,and because (b) replacement of the Pmn aminegroup with a less acidic OH group changes theslope to ∼1 and gives a pH-rate profile with asingle pKa of 7.5 (16). It is unclear if the ap-parent pKa of 7.5 reflects an important mech-anistic feature of the reaction with full-lengthaminoacyl tRNA or manifests as a consequenceof important tRNA-rRNA interactions missingin the Pmn reaction.

To resolve some of these ambiguities,Bieling et al. (14) used quench-flow methods tomonitor the pH dependency using aminoacyltRNAs, whereby the α-amine was substitutedwith the less reactive OH group, and they wereable to measure rates where the chemical stepwas rate limiting. In contrast to the reactionwith Pmn, the measured rate with the modifiedaminoacyl tRNA showed no dependency onpH, providing no evidence for acid-base catal-ysis in the peptidyltransferase reaction. Trobro& Aqvist (46) suggested that the pKa observedin the Pmn reaction reflects deprotonationof the N terminus of a ribosomal protein,L-27, important for A-site substrate bindingin the presence of Pmn. According to theirsimulations, the absence of the A76 phosphategroup in Pmn decreases the pKa of the L27N terminus and becomes pH titratable. Incontrast, with full-length aminoacyl tRNAor C-Pmn, the presence of the A-site A76phosphate favors protonation of the L27 N ter-minus, which, in turn, accounts for the absenceof an observed pH dependency (14, 15).

5. INDUCED-FIT ANDSUBSTRATE-ASSISTEDCATALYSIS

In vitro the 50S subunit can synthesize peptidebonds as rapidly as the entire 70S ribosome can(47), indicating that structural and mechanisticfeatures of the peptidyltransferase reactionlikely hold for both forms of the catalyst.High-resolution crystal structures of theH. marismortui 50S subunit complexed withfull-length or minimal A-site and P-sitesubstrates, TSAs of various lengths andcomposition, and products reveal a PTCcomposed entirely of RNA with no proteinatoms within 15 A of the reaction center (32,48–51). These structures, supported by earlybiochemical data, implicate rRNA rather thanprotein as the key component in the catalysis ofpeptidyl transfer (52, 53). The A- and P-loopregions of 23S rRNA interact with the univer-sally conserved 3′-terminal CCA residues ofthe A- and P-site tRNAs, respectively, holdingthem in place and properly orienting themfor peptidyl transfer. 23S rRNA nucleotidesC2063, G2447, A2450, A2451, C2452, U2506,G2583, U2584, U2585, A2602, and A2606 re-side near or at the heart of the PTC and appearto play important roles in catalysis (49, 54, 55).The PTC adopts the same global fold in the50S subunit of H. marismortui complexed withvarious ligands (32, 49, 50, 55, 56), the 50S ofDeinococcus radiodurans (57), the 70S ribosomeof Thermus thermophilus (51, 58, 59), andvacant 70S ribosomes of E. coli (60). However,within these structures, particular active-sitenucleotides, such as A2602, adopt differentorientations depending upon the occupancyand identity of the A- and P-site substrates (49).

Biochemical and structural data suggestthat 23S rRNA residues closest to the activesite (C2063, A2451, U2506, U2585 andA2602) play a pivotal role in the structuralorganization of the PTC (40, 61). With anempty A site, the P-site substrate, a C74, C75,or A76 oligonucleotide linked to an analog of agrowing peptide chain, adopts a conformationunfavorable for reaction. C2063 and A2451


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reside on one side of the carbonyl carbon andU2585 on the other side, sterically shieldingthe reaction center from nucleophilic attackand apparently preventing premature hydrol-ysis. This conformation remains unchangedwhen the A site contains Ch-Pmn, Pmn witha C corresponding to tRNA C75, and an OHgroup replacing the α-amine. In this ternarycomplex, the Ch-Pmn OH group points towardthe carbonyl oxygen, precluding access to thecarbonyl carbon (49). However, the inclusionof just C74 to the A-site substrate, CCh-Pmn,triggers rearrangement of active-site residuesU2506, G2583, and U2585 (49). The resultinginduced conformation positions the P-sitepeptidyl tRNA closer to the center of the activesite and causes a rotation of the peptidyl-tRNAcarbonyl carbon to an orientation appropriatefor nucleophilic attack (Figure 4b).

When 50S or 70S structures revealed nometal ions close enough to the active site to pro-mote catalysis (51, 55), extensive mutagenesisof active-site residues A2451, U2506, U2585,and A2602 (16, 34, 38–40, 61), the adjacentG2447 residue (38, 39, 42), and the noncanon-ical A2450-C2063 base pair (34, 62) began inearnest, with the aim of identifying possibledirect roles for the rRNA itself in catalysis.With Pmn as the A-site substrate and Met-Phe-tRNAPhe as the P-site substrate, A2541,U2506, U2585, and A2602 mutations severelyimpaired the rate of peptide bond formation(a 30–9,400-fold rate reduction when com-pared to wild-type ribosome) (16, 40, 42, 61).Unexpectedly, despite the broad range of sub-stantial energetic effects, none of the singlemutations affected the rate of peptide bond

formation with either full-length aminoacyltRNA or even with just C-Pmn as the A-sitesubstrate (40, 42, 61), suggesting these sub-strates mask or somehow offset the deleteri-ous effects observed in the Pmn reaction. Mostlikely, these mutations disrupt positioning ofPmn and its nucleophilic nitrogen in the activesite, but the additional interactions provided byC-Pmn or tRNA can overcome these effectsby stabilizing a more catalytically active state(15, 40).

In contrast, mutation of the A2450-C2063wobble base pair to G2450-U2063 decreasedthe peptide bond formation rate ∼200-foldeven with full-length aminoacyl tRNA (34).Perhaps G2450-U2063 fails to form the iso-steric wobble pair or lacks the electrostatic fea-tures of the AC wobble, which may becomeprotonated to achieve hydrogen bonding com-plementarily (34). Chirkova et al. (63) appliedatomic mutagenesis to study A2450-C2063 andshowed that disruption of the wobble pair hadlimited effects on peptidyl transfer when usingPmn but markedly impaired protein synthesisin translation assays. The authors hypothesizedthat the A2450-C2063 base pair may not con-tribute to the peptidyltransferase reaction butmay have significance for communication be-tween the PTC and the nascent polypeptide inthe exit tunnel, which, in turn, may regulate theefficiency of tRNA translocation.

Crystal structures (55) and molecular dy-namics (MD) simulations (64, 65) of theinduced state of the 50S subunit suggestthat active-site residues and ordered watermolecules engage in an intricate hydrogenbond network (Figure 5). Water molecules

−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−→Figure 5Peptidyltransferase center (PTC) catalytic mechanisms. (a) Proposed concerted six-membered ring proton shuttle mechanism. Theamine nucleophile attacks the carbonyl center and proton shuttling, via the 2′ hydroxyl group of A76, resulting in the formation of thenew peptide bond. (b) Proposed eight-membered ring double-proton shuttle mechanism. Similar to the six-membered ring mechanism,the amine nucleophile attacks the carbonyl center; however, a double-proton shuttle occurs via an active-site water molecule and the 2′hydroxyl group of A76. This active-site water molecule is positioned by hydrogen bonds between the 2′ and 3′ positions of A76.(c) Proposed hydrolysis reaction mechanism catalyzed by the ribosome. The P-site peptide is released when the nucleophilic watermolecule, which is orientated by hydrogen bonds with the RF GGQmethyl motif, A76 of the P-site tRNA, and A2451 of the ribosome,attacks the carbonyl center. Figure 5a adapted from Trobro & Aqvist (64), Figure 5b adapted from Wallin & Aqvist (77), andFigure 5c adapted from Trobro & Aqvist (109) and Jin et al. (124).

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H

H

H

a b

c N

NN

N

NH2

NH2

NH2

O

OO

O

C75

O

PeptideH

O

H

O

N

N

OCH3

H

N

N

NN

N

H

H

H

OH

H

A76

A2602

RF Glnmethyl

NN

O

C2452

N

N

N

N

O

HO

H

A2451

O

H

O C C2063

A76

O

OO

tRNA

O

Peptide NH

H

R

H

H

O

H

C2063

2'OH

HO'2 A2451

tRNA

O

H

O

H

U2584

2'OH

A2602 NH2

O

H

O C C2063

A76

O

OO

tRNA

O

PeptideN

H

H

R

H

H

O

H

C2063

2'OH

HO'2 A2451

tRNA

O

H

O

H

U2584

2'OH

A2602 NH2

O

H

O C C2063

A76

O

OOH

tRNA

O

Peptide N

H

H

R

H

O

H

C2063

2'OH

HO'2 A2451

tRNA

O

H

O

H

U2584

2'OH

A2602 NH2

A76

O

OO

tRNA

O

Peptide NH

H

R

H

H

O

H

H

O

H

C2063

2'OH

O C C2063

HO'2 A2451

tRNA

O

H

O

H

U2584

2'OH

A2602 NH2

A76

O

OO

tRNA

O

Peptide NH

H

R

H

H

O

H

H

O

H

C2063

2'OH

O C C2063

HO'2 A2451

tRNA

O

H

O

H

U2584

2'OH

A2602 NH2

A76

O

OO

tRNA

O

Peptide NH

H

R

H

H

O

H

H

O

H

C2063

2'OH

O C C2063

HO'2 A2451

tRNA

O

H

O

H

U2584

2'OH

A2602 NH2

P-site substrate

A-site substrate/attacking water molecule

Ribosome

Ordered water molecules in PTC hydrogen bond network

Water molecule proposed to stabilize charge


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positioned by active-site residues may aid catal-ysis by stabilizing the tetrahedral intermedi-ate(s) (see Figure 5) and facilitating protontransfer to the carbonyl oxygen or the leavinggroup (Figure 5b). Although mutagenesis workhas not identified direct roles for rRNA in catal-ysis, one functional group on the P-site peptidyltRNA has emerged as a critical element in thereaction mechanism, the A76 2′ OH (66). Instructures of the 50S complexed with differentTSAs, the hydrogen bond between the N3 ofA2451 and the α-amine observed in the pre-reaction state crystal structure (32) no longerpersists, but the A76 2′ OH of the P-sitepeptidy-tRNA (55) resides within hydrogenbonding distance of the α-amine, the A2452 2′

OH, and an ordered water molecule.Atomic mutagenesis and model studies sup-

port the functional relevance of these interac-tions. Mutation of the A2451 2′ OH impairspeptidyltransferase activity by about 10-fold(67). Using full-length aminoacyl tRNAs con-taining either 2′-H- or 2′-F-substituted A76,Weinger et al. (66) observed a rate reduc-tion of at least 106-fold, below the detectablelimit. The authors hypothesized that A76 2′

OH may help position the nucleophile, act asa general base to deprotonate the nucleophile,and/or act as a general acid to protonate theremaining A76 3′ O as the tetrahedral inter-mediate resolves (66). Examining intramolec-ular aminolysis from an acylated nucleoside inorganic solvent, Bayryamov et al. (68) showedthat acyl group transfer proceeds 3,600-foldfaster from adenosine than from deoxyadeno-sine. Minimally, these results demonstrate thata cis-oriented vicinal OH group can facilitateacyl transfer from the adjacent 3′ O as proposedfor the ribosomal peptidyltransferase reaction.These observations notwithstanding, Koch andcolleagues (69) reported evidence that the P-site A76 2′ OH is dispensable in a cell lysatetranslation assay. The basis for these apparentlyconflicting observations remains unclear.

The apparent importance of the 2′ OH ledto the proposal of substrate-assisted catalysis,whereby a functional group on the substratecontributes to catalysis. Several protein

enzymes employ this mechanism, includingGTPases, serine proteases, type II restrictionendonucleases, lysozyme, and hexose-1-phosphate uridylyltransferase (70). Althoughthe dramatic 106-fold reduction of the pep-tidyltransferase activity caused by removal ofP-site A76 2′ OH has firmly established animportant functional role for this group, somehave presented alternative views on whether the2′ OH functions in terms of “substrate-assistedcatalysis.” For example, Wolfenden ascribessubstrate-assisted catalysis to interactions be-tween substrate and catalyst that enhance therate relative to the uncatalyzed. As suggestedexperimentally and computationally (65, 71–74), the 2′-OH group may simply supplant therole that water molecules play in model esteraminolysis reactions in aqueous solution. Thecontribution from the 2′-OH group thereforemay not exceed the corresponding contri-bution from water in the solution reactionbut becomes essential for reaction within thespecial context of the ribosome.

Additional support for the functional rel-evance of the crystal structures comes fromthe observation that the ribosome exhibits adiastereoselective preference in TSA binding.Structures of the H. marismortui 50S subunitwhen complexed with TSAs of different ste-reochemistry (Figure 1d ) (55) suggest that thetetrahedral intermediate(s) likely has (have) (S)chirality. If the TS has an (S) chirality, the crit-ical P-site A76 2′ OH sits near the attackingα-amine, whereas in the (R) configured TS, the2′ OH sits closer to the universally conservedA2451. A study tested binding to the ribosomefor two TSAs with (S) and (R) chirality at thetetrahedral center. The TSA with (S) chiral-ity bound with an affinity of ∼77 nM, whereasthe (R) chirality diastereomer bound with a 20-fold weaker affinity of ∼1,500 nM (75), consis-tent with an (S) chirality TS implicated by theproton shuttle mechanisms. Nevertheless, theribosome’s 20-fold diastereoselectivity in TSAbinding appears modest in comparison to the106-fold rate acceleration, reminding us thatTSAs generally provide only crude mimics ofa reaction’s true TS.

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In silico simulations of the ribosome haveled to a significant number of mechanistichypotheses. In evaluating these hypotheses,one needs to be mindful of the important initialparameters used in these simulations (reviewedin Reference 76), including atomic-levelaccuracy, assignment of solvent atoms (water,cations, and anions), and functional relevanceof the structural data used as the starting point.Unfortunately, X-ray data, especially lower-resolution structures, rarely provide directevidence for factors that may influence thesimulation, prohibiting precise detection ofsteric clashes and/or inducing nonfunctionalstructural transitions. Naturally, the accuracyof the empirical equation that governs theintermolecular forces and the evaluation ofits terms dictate the overall outcome of thesimulation; even the most precisely developedset of parameters cannot account realisticallyfor the dynamics of these systems. Thesecaveats notwithstanding, the computationalresults can provide valuable insights that formthe basis of experimentally testable hypotheses.

Trobro & Aqvist (64, 65) and Wallin& Aqvist (77) used MD simulations andfree-energy perturbation simulations in com-bination with an empirical valence-bonddescription of the reaction energy surface toevaluate possible catalytic mechanisms. Com-putational analysis of the reactants and tetra-hedral intermediate states of the PTC showedthat the ternary complex, including associatedwater molecules, does not need to undergosignificant rearrangement during peptidyltransfer, suggesting that the hydrogen bondnetwork formed through the induced-fit mech-anism preorganizes the active site for catalysis(Figure 5). The simulations further suggestthat the PTC achieves catalysis by reducingthe entropic energy of activation rather thanby providing general acid-base catalysis (64)(see Section 6). In this model, A76 2′ OHof the P-site bound tRNA acts as a protonshuttle that bridges the attacking α-amineand the leaving 3′-oxygen through a six- (64,65) or eight-membered ring TS (77). In thesix-membered ring TS, the α-amine attacks

the carbonyl carbon to form the tetrahedralTS. The TS resolves by transferring a protonto the A76 2′ OH, which simultaneouslydonates its proton to the 3′-oxygen leavinggroup, thereby activating the intermediate forexpulsion of the leaving group (Figure 5a).

The eight-membered ring proposal de-rives from the observation by Schmeing andcolleagues (55) that in the structure of the 50S-TSA complex (Figure 1d ) a water moleculeresides within hydrogen bonding distance ofthe P-site A76 2′ and 3′ oxygen atoms and thecarbonyl group of C2063 (Figure 5b). In theeight-membered ring TS, the α-amine attacksthe carbonyl carbon, and the tetrahedral TS re-solves via a proton shuttling from the α-amineto the 2′ OH and from the 2′ OH to the 3′-Oleaving group via a properly positioned watermolecule (Figure 5b). Another active-sitewater molecule, held in place by A2602 andU2584, presumably stabilizes the tetrahedralintermediate via interactions with the oxyanion(Figure 5a,b). Computational analysis predictsthat this water-mediated stabilization of thetetrahedral oxyanion intermediate holds forboth six- or eight-membered ring TSs.

23S rRNA residues participating in the pro-posed hydrogen bond network (C2063, A2451,and A2602) have been studied; in these stud-ies, nucleotides were mutated to C3 linkers andabasic residues, and 2′ OH groups were mutatedto 2′H, 2′F, 2′OCH3, and 2′NH2 (41, 67, 78,79). However, all the reactions were done usingPmn as the A-site substrate, which, as describedabove, responds differently to active-site muta-tions than does C-Pmn or CC-Pmn. Therefore,additional experiments are required to test thecurrently proposed mechanistic model, such asaccessing the catalytic activity of the mutantsusing C-Pmn, CC-Pmn, or full-length tRNAsubstrates and detecting water interactions us-ing Fourier transform infrared spectroscopy(reviewed in Reference 80) of ribosomes withatomic mutations at important residues (basedon Figure 5b).

Recent high-level quantum chemical abinitio calculations analyzed the six- and eight-membered TSs and calculated their energies


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and kinetic isotope effects (77). The calcula-tions suggest that the six- and eight-memberedring reactions occur with late TSs, wherebythe attacking amine bears little charge, and theC-O bond cleavage has advanced significantly.Between the two proposed mechanisms,the calculated activation enthalpy for theeight-membered “double proton shuttle”mechanism more closely agrees with theexperimentally measured activation enthalpy,possibly supporting the functional relevanceof the crystal structures at the active site.With the exception of the nucleophile isotopeeffect, the calculated kinetic isotope effectsfor this mechanism qualitatively match thosereported for the uncatalyzed acylation of ani-line and hydrazinolysis of methyl formate (seeSection 3), suggesting that the PTC and uncat-alyzed reactions follow analogous mechanisticpathways. Overall, most biochemical data—including the absence of any deleterious singlemutations in the active site, the absolute impor-tance of the P-site A76 2′ OH, the entropic andenthalpic contributions to the activation barrier(81), high-resolution structural data, and com-putational analysis (77, 82)—give support to theproposed double proton shuttle mechanism.

The detailed structural analysis of ribosomescomplexed with different substrate analogsmimicking different stages of the reaction hasrevealed both the precise alignment of theA- and P-site substrates through interactionsbetween the conserved 3′ CCA sequences andthe A- and P-loop elements of the 23S rRNA,and the alignment of the nucleophilic α-amineof the A-site substrate within the active site (49,55, 83–85). Computational analysis of avail-able crystal structures, mutation of the A76 2′

OH, and entropic and enthalpic energies of ac-tivation favor a mechanism that involves theP-site A76 2′ OH and a water molecule po-sitioned between the P-site A76 2′- and 3′-Omediating a concurrent eight-membered dou-ble proton shuttle (55, 64, 77). Ribosomalresidues do not appear to engage in cataly-sis directly but undergo crucial active-site con-formational changes required for catalysis (14,15, 38–40, 42). In addition to positioning the

substrates in an orientation optimal for reac-tion, active-site 23S rRNA and water moleculesmay contribute to catalysis in the followingways: (a) by providing a preorganized envi-ronment with spatial and electrostatic comple-mentarity to the highly polar tetrahedral TS,thereby abrogating the need for solvent reor-ganization along the reaction trajectory; (b) byshielding the peptidyl tRNA from prematurehydrolysis; and (c) by aiding the expulsion of theleaving group by positioning functional groupsor active-site water molecules that may helpneutralize the developing charge.

6. ENTROPIC AND ENTHALPICACTIVATION OF THE PEPTIDYLTRANSFER REACTION

TS theory provides a general framework foranalyzing enzymatic reactions by relatingthe rate of a reaction to the difference infree energy between the TS and the groundstate. Assuming a thermodynamic equilibriumbetween the ground state and TS, the theoryallows calculation of the standard Gibbs energyof activation (�G‡) for a particular reaction(86). Determination of the reaction rate inthe presence and absence of an enzyme as afunction of temperature allows for delineationof the relative free-energy barrier of activationinto enthalpic (�H‡) and entropic (T�S‡)components and can provide insights into thecatalytic mechanism (87).

In enzymatic reactions involving twosubstrates, the entropy term significantly influ-ences the activation free energy. Work by Page& Jencks (88) supported a view in which for-mation of the enzyme-substrate complex servesas an “entropy trap” to juxtapose and orientthe reacting groups. Substrate binding forcesoffset the energetic cost associated with loss ofrotational and translational entropy, therebyenhancing the enzymatic reaction relative to thenonenzymatic reaction (89). However, in addi-tion to the loss of rotational and translationaldegrees of freedom associated with substrates,the entropy of activation involves thermody-namic changes in the entire system, including

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enzyme, solvent, and other system components(64). Indeed, recent quantitative estimates ofthe activation entropy for enzymatic reactionshave suggested that substrate positioningimparts less rate enhancement than previouslythought and that the free energy associated withsolvent reorganization along the reaction coor-dinate plays a considerable role (64, 65, 73, 90).

After binding their substrate(s), enzymesmay enhance the rate of a reaction further as achemical catalyst, using additional forces of at-traction to stabilize chemical intermediates thatapproach the TS. In reactions involving gen-eral acid-base catalysis, enzymes typically pro-duce a major reduction in the enthalpy of ac-tivation, consistent with the formation of newhydrogen bonds and electrostatic interactionsin the TS (91). If the ribosome utilizes directchemical catalysis, the rate enhancement wouldbe expected to have a pronounced enthalpicsignature.

Using a structural model derived fromsuperimposed H. marismortui 50S subunitcrystal structures, Moore and colleagues (56)proposed that the ribosome’s acceleration ratederives exclusively from attenuation of the ac-tivation entropy by reducing translational androtational degrees of freedom associated withthe substrates. In their model, correct position-ing of the reactants is achieved via interactionsbetween the A- and P-site tRNAs 3′ ends andthe A and P loops and interactions involving theattacking α-amine with the N3 of A2451 andeither the 3′ terminal 2′ OH of the P-site tRNA

or the 2′ OH of A2451. The α-amine group ispositioned adjacent to the carbonyl carbon ofesterified P-site tRNA in an orientation suitablefor a nucleophilic attack. Corroborating thesestructural data, biochemical (85) and in vitrogenetic studies (84) have demonstrated directbase-pairing interactions between the tRNA 3′

CCA ends and the 23S rRNA A and P loops.Sievers et al. (12) reported thermodynamic

parameters for the ribosome-catalyzed reactionand for two uncatalyzed reactions (Table 1).Consistent with the substrate alignment idea,they showed that the activation enthalpy im-poses a 6.9 and 8.2 kcal/mol greater energeticpenalty in the ribosome-catalyzed reactioncompared to two solution reactions (12, 74),supporting mutagenesis studies that argueagainst a significant role for general acid-basecatalysis. A higher �H‡ for the ribosome-catalyzed reaction may reflect binding interac-tions that weaken en route to the TS. Alterna-tively, by excluding water from the PTC, theribosome may inhibit formation of favor-able solvent interactions that stabilize theTS in the uncatalyzed reaction. The differ-ence in the activation entropy [�(T�S‡) =−17.7 kcal/mol] and activation enthalpy(��H‡ = 8.2 kcal/mol) between the ribosome-catalyzed reaction and the solution reactionproduces the ��G‡ of −9.5 kcal/mol, whichsufficiently accounts for the observed >107

rate enhancement (74). The finding that the�(T�S‡) term is the major contributor to��G‡ led to the description of the ribosome

Table 1 Comparison of rates and thermodynamic activation parameters for solution and ribosome-catalyzed reactions

Reaction (Reference) Amine Ester Rate�G‡

(kcal/mol)�H‡

(kcal/mol)T�S‡

(kcal/mol)Solution (12) Tris fGly-ethylene glycol 3 × 10−4a 22.2 9.1 −13.1Solution (74) Glycinamide fPhe-TFE 3 × 10−5a 23.5 7.8 −15.7E. coli ribosome (12) Puromycin fMetPhe-tRNAfPhe 103a,b 14.0 16 2.0T. thermophilis ribosome (93) Puromycin fMet-tRNAfMet 120c 17.4 22.2 4.8E. coli ribosome (92) Phe-tRNA fMet-tRNAfMet 130c 15.0 17 2.0

aM−1s−1.bSubsaturating.cs−1.


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as an entropy trap (12, 74). Reactions withfull-length aminoacyl tRNAs (92) and withribosomes from thermophilic bacteria (93)gave similar thermodynamic parameters.

Sievers et al. (12) suggested that theobserved entropic contribution to the rateacceleration could also reflect changes insolvation entropy rather than the exclusivereduction of the substrate translational androtational entropies. For reactions in aqueoussolution, contributions from the free energy ofsolvation complicate the interpretation of �H‡

and T�S‡ terms (94). For example, formationof the zwitterionic intermediate in esteraminolysis might involve solvent reorganiza-tion, rendering the entropy of activation lessfavorable. Formation of this intermediate in awater-poor environment, such as the preorga-nized active site of the ribosome, would incurno such penalty from solvent reorganization.

Trobro & Aqvist (64) performed MDsimulations on a superimposed 50S model toexamine possible catalytic mechanisms andestimate activation enthalpy and entropy forribosome-catalyzed peptide bond formation.The computational analysis showed agreementwith kinetic measurements regarding themagnitude of �G‡ and attributed ribosomecatalysis almost exclusively to elimination ofthe entropic penalty associated with solventreorganization. In these simulations, thereaction in bulk water requires a significantreorganization of water molecules owing tocharge separation involved in formation ofthe TI± . In contrast, the ribosome active siteprovides a preorganized hydrogen bond net-work that stabilizes the TS and intermediatesalong the reaction path, thereby precludingthe need for solvent reorganization (seeFigure 3 and Figure 5a,b for possible mech-anistic pathways). Moreover, the simulationsestimate a relatively small free-energy contri-bution from substrate positioning in the groundstate ternary complex (<1 kcal/mol). Subse-quent MD simulations on 50S ribosome withbound TSAs yielded similar results (55, 65).

Quantum mechanics–based computationalapproaches using the 50S model led Sharma

et al. (73) to similar conclusions. For twononenzymatic reference reactions (ammoniaattack on methylmethanoate and methylamineattack on a ribosyl ester), quantum mechanicsresults attribute a significant fraction of the ac-tivation entropy to change in solvation entropy(3 kcal/mol for solvation entropy and 2 kcal/molfor reactant orientational entropy) (73). To ex-plore the origin of the overall catalytic effect(��G‡), they examined the electrostatic inter-actions between the PTC and the substratesin the ground state and the TS. These cal-culations indicate that active-site electrostaticfree energy (�Gele) is greatly reduced in theTS compared to the ground state [��Gele =�Gele(TS) − �Gele(GS) = – 8 kcal/mol]. Con-sistent with the idea that in the ground state theactive-site dipoles are already partially orientedtoward the TS charge center, Sharma et al. (73)attributed approximately –4 kcal/mol ��Gele

to active-site dipole preorganization. Further-more, for atoms of the substrate that undergocharge changes upon moving from the groundstate to the TS, �Gele stabilization is greater inthe ribosome-catalyzed reaction than in the so-lution reaction. The authors conclude that theribosome catalyzes peptidyl transfer by lower-ing the electrostatic free energy barrier of thereacting system.

Recently Trobro & Aqvist (77) performedhigh-level quantum chemical calculations onthe PTC model that emerged from previousMD simulations. They computed enthalpic andentropic contributions from an interaction be-tween the substrate oxyanion and the watermolecule positioned by A2602 and U2584 (55).This water molecule is distinct from the wa-ter molecule proposed to engage in the shut-tling of a proton from the α-amine group to theleaving group (Figure 5b). According to theirmodel, inclusion of a single water moleculeyields a small activation entropy, whereas a sec-ond water molecule causes a 3 kcal/mol in-crease in T�S‡. These oxyanion-screening wa-ter molecules had an opposite effect on theactivation enthalpy: A single water moleculematched the experimentally determined ac-tivation enthalpy [17 kcal/mol (12)], and

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addition of a second water molecule decreasedthe enthalpy barrier to a value similar to the so-lution reaction. Deletion of the water moleculesaltogether increased the activation enthalpyby 3 kcal/mol. Therefore, in the ribosome-catalyzed reaction, oxyanion screening by a sin-gle water molecule may provide the optimal bal-ance between the entropic cost of preorganizingthe water molecules and the favorable decreasein the enthalpic term.

The strategy employed by the ribosome toaccelerate the rate of peptide bond formationrelative to the uncatalyzed reaction attenuatesthe entropic penalty associated with reachingthe TS. While the solution reaction has anunfavorable activation entropy, for the ribo-some reaction the decrease in T�S‡ approaches14 kcal/mol, with substrate positioning and sol-vent reorganization as two potential sourcesof this effect. Although the relative contribu-tions remain unknown, the current experimen-tal evidence supports the view that the ribosomegains significant rate enhancement by organiz-ing the PTC so as to eliminate the need for sol-vent reorganization along the reaction coordi-nate. Figures 3 and 5 include possible strategiesto accomplish proton transfer without solventreorganization.

7. PEPTIDE RELEASE

In all organisms, one of three nearly conservedstop codons signals the end of translation whenpresent in the A site of ribosome. However, stopcodons are decoded by RF proteins that trig-ger a hydrolysis reaction in the ribosome activesite, resulting in the release of the polypeptidechain from the P-site tRNA. Despite the dis-covery of RFs more than 30 years ago (95), ourunderstanding of their roles in translation ter-mination remains less developed compared toother aspects of translation.

Efficient termination requires two proteinfactors: class I and II RFs. Class I RFs directlyrecognize the stop codon and participate in cat-alyzing the hydrolysis reaction. Bacteria havetwo class I RFs (RF1 and RF2) that decodethe three stop codons (UAG, UAA, and UGA)

with overlapping specificities (96). Eukaryotesand archea have one omnipotent class I RF(eRF1), which decodes all three stop codons(97). Because both RFs and aminoacyl tRNAsinteract with the same ribosomal structural el-ements, RFs have been hypothesized to act asfunctional tRNA mimics (98). Class II RFs areGTPases with different functions in bacteriaand eukaryotes.

In bacteria, the class II RF (RF3) initiatesthe dissociation of the class I RF from the post-termination ribosomal complex after peptidyl-tRNA hydrolysis (99). The eukaryotic class IIRF (eRF3) associates with eRF1 in the cyto-plasm and interacts with the preterminationribosomal complex to induce conformationalchanges (100).

The study of peptide release has been chal-lenging owing to the lack of detailed kineticcharacterization of the reaction pathway, andstructures of isolated RFs are different fromribosome-bound states (101, 102). However,recent high-resolution 70S structures with RF1(103, 104) or RF2 (104–106) in the A site,deacylated-tRNA in the P and E sites (103,104, 107), and computational analysis (108–110) have provided valuable insights into thecatalytic mechanism of peptide release.

7.1. Catalysis of Peptide Release

At the end of translation elongation, the Cterminus of the polypeptide chain attached tothe P-site tRNA undergoes nucleophilic at-tack at the ester carbon by a water molecule,which leads to release of the newly synthesizedpolypeptide. In many respects, the mechanismof peptide release resembles the mechanism ofpeptide bond formation, differing only in theidentity of the nucleophile (water versus amine)and the requirement for accessory protein fac-tors in peptide release. Both reactions occur inthe same active site and begin with the sameground state complex, where the labile P-siteester linkage sits in the PTC.

Structural features of this ground state (seeSections 3, 5, and 6) protect the P-site peptidyl-tRNA ester linkage from premature hydrolysis


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in the absence of an A-site substrate (seeSection 5). Similarities between peptidyl-transfer and peptidyl-release reactions and theproposed functional mimicry between deacy-lated tRNA and class I RFs raise the possibilitythat binding of class I RFs and A-site deacy-lated tRNA may induce similar conformationalchanges to activate the ribosome for hydroly-sis of the peptidyl tRNA. Binding of deacylatedtRNA in the A site results in a large (up to 300-fold) increase in the peptidyl-hydrolysis rate.Interactions between the 23S rRNA A loop andthe A-site tRNA 3′ end may possibly induce acatalytically active state (111–113), as suggestedfor the peptidyltransferase reaction (Section 5).The induced-fit component contributes 2×105

in release catalysis (112); however, the mecha-nistic details of this reaction remain unclear.

Recent mutational and computational stud-ies have revealed potential contributions tocatalysis of peptide release by class I RFs. Be-cause water is a weaker nucleophile than anamine, peptide release might require greatercatalysis than peptide bond formation. Unlikethe attacking α-amine, which gains a catalyticadvantage from being tethered to the tRNA,the nucleophilic water molecule gains no suchadvantage from the RF. Possibly, precise pack-ing of the active site helps orient the nucle-ophilic water properly for reaction. In sup-port of this idea, mutation of PTC residueshad deleterious effects on peptide release (upto 300-fold in the case of A2602 mutations),whereas the same mutations had only mini-mal effects on the peptidyltransferase reaction(40, 114, 115).

Frolova et al. (116) predicted that theGGQmethyl motif (Qmethyl represents N5-methylglutamine) within the RF interacts withthe PTC in a manner analogous to the 3′

CCA ends of tRNAs. Subsequent biochemical(112, 113, 116–119) and structural studies(101, 103, 104, 106, 119) have supported thisprediction. An early mechanistic model positedthat the conserved Glnmethyl helps position theattacking water molecule (101). Although mu-tation of the Glnmethyl to alanine or tryptophanonly had minimal decreases in activity (112,

113, 117), mutation of the Glnmethyl to as-paragine or aspartic acid had more severeeffects on activity (a 7,600- and 9,500-folddecrease, respectively), consistent with animportant role in catalysis (99). Additionally,mutation of either of the two flanking glycines(Gly) dramatically reduced the rate of peptiderelease (112, 113). Nucleophile partitioningexperiments indicated that Glnmethyl plays arole in excluding other potential nucleophilesfrom the active site (112), supporting the ideathat precise packing of the catalytic center maycontribute to optimal RF function. Extensivemutational analysis of other conserved residuesin E. coli RF1 failed to identify other func-tionally important residues (112). Recently,T. thermophilus 70S structures have revealeddetails of the product state of the terminationcomplex containing RF1 (103) or RF2 (106,119), deacylated tRNAs in the P and E sites, anda messenger RNA containing UAA or UGAstop codons. In these structures, the GGQmethyl

motif interacts with PTC residues and with A76of the P-site tRNA. The backbone amide (NH)of the Glnmethyl lies within hydrogen bondingdistance of the deacylated P-site tRNA 3′ OH,suggesting that the residue might facilitatehydrolysis by hydrogen bonding to the leavinggroup (103, 106, 119). This backbone amidealso may stabilize the tetrahedral oxyanionintermediate, as superimposition of a TSA ontothe structure followed by computational anal-ysis position the two groups within hydrogenbonding distance via a bridging water molecule(see Figure 5c) (103, 106, 119). Additionally,the backbone amide directly contacts andpresumably properly orientates the attackingwater molecule (see Figure 5c) (103, 106,119). The side chain amide of Glnmethyl pointsaway from the site of catalysis toward a pocketformed by A2451, C2452, U2506, and theA76 ribose moiety of the P-site tRNA. Theconformational flexibility of the two adjacentGly residues allows the proper orientation ofGlnmethyl, possibly explaining the deleteriouseffect of Gly mutations (103). To assess the roleof the backbone amide group, Korostelev et al.(119) mutated the RF1 Glnmethyl to proline

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and observed stoichiometric codon-dependentbinding, but no detectable hydrolysis activity.Computational modeling of the terminationcomplex with this mutant RF1 gave no indi-cation that proline substitution would induceany significant changes in the RF1 and thesurrounding PTC conformations (119). Theloss of hydrolysis activity but not bindingsuggests that the Glnmethyl plays an importantrole in catalysis.

MD simulations (Figure 5c) similar tothose used in Section 5 for peptidyl transferhave suggested a model for catalysis of peptiderelease involving an active-site hydrogen bond-ing network that includes the Glnmethyl residue.In this model, the side chain NCH3 groupdonates a hydrogen bond to the nucleobasecarbonyl group of C2452, and the side chaincarbonyl group accepts a hydrogen bond fromthe nucleophilic water molecule, positioningit for nucleophilic attack (109). The Glnmethyl

backbone NH and carbonyl groups formhydrogen bonds to A2602 and the bridgingwater molecule, which helps stabilize the tetra-hedral intermediate (109). According to thecalculations, the posttranslational methylationof the Gln side chain contributes to catalysisby restricting the mobility of Glnmethyl, therebystabilizing the hydrogen bond network (109).Glnmethyl mutants remain relatively active;apparently because in the absence of the sidechain methylcarboxamide group, these mutantscan accommodate an extra water moleculethat interacts with the protein backbone andsupplants the role of the side chain (109). Thecomputational model further suggests thatthe two Gly residues of the GGQmethyl motifinteract with A2602, consistent with the experi-mentally observed deleterious effect on peptiderelease from the mutation at A2602 (109). Thecalculations predict that the RF accelerates thehydrolysis rate by a factor of ∼105 (109), whichcorresponds well to the measured spontaneousand RF-induced hydrolysis rates of 2×10−5 s−1

and ∼1 s−1, respectively (112, 113).The mechanistic model for peptide release

proposed from structural and computationalanalysis posits that the binding of RFs to the A

site induces conformational changes in active-site ribosomal nucleotides analogous to thosefor the peptidyltransferase reaction, enablingcatalysis of peptide release. The universallyconserved GGQmethyl motif resides near thePTC and engages in a network of hydrogenbond interactions in which the Glnmethyl maystabilize the TS via its backbone amide and po-sition the attacking water nucleophile in thecontext of a tightly organized pocket, formedby 23S rRNA, P-site tRNA nucleotides, andthe RF itself. Like the PT reaction, the 2′ OHof A76 of the P-site peptidyl tRNA likely par-ticipates in shuttling a proton from the attack-ing water molecule to either the leaving groupor the carbonyl oxygen. In agreement with thisshuttling role, deletion of this 2′ OH nearlyabolishes the peptide release activity of E. coliRF1 (120).

8. CONCLUSION AND OUTLOOK

A decade ago we had little knowledge of whatthe ribosome’s PTC looked like or how its com-ponents synergize to facilitate the synthesis ofproteins. Through significant advances in ribo-some structural biology, the availability of sub-strate analogs, the development of functionalassays and computational approaches, and theability to mutate the ribosome with atomic-level precision, the understanding of PTC-mediated catalysis has made great strides. Wenow know that the reaction center consists en-tirely of RNA and that peptidyl transfer from aP-site substrate to an A-site substrate requires aconformational change to activate the ribosomefor catalysis. The reaction pathway likely fol-lows that for classic aminolysis of a carboxylateester: First, there is a nucleophile attack at thecarbonyl carbon to form a TI± ; and followingloss of a proton from the nucleophilic nitrogen,the intermediate collapses to expel the leavinggroup and form the amide (peptide) bond. Keyquestions regarding how the ribosome catalyzesthis reaction pertain to the nature and timing ofproton removal from nitrogen and the strategyfor facilitating leaving-group expulsion. Theprevailing view implicates the A76 2′-hydroxyl


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group of the P-site tRNA as a critical elementin both of these roles, serving to “shuttle” aproton from the amine to the leaving groupeither directly or via a water molecule.The absolute requirement for the 2′-hydroxylgroup during peptidyl transfer supports thisview.

Further insights about the catalytic strate-gies used by the ribosome come from analysisof the activation barriers for the nonenzymaticand PTC catalyzed reactions. The observationthat, in the ribosome’s PTC, ester aminolysis,occurs with a much smaller entropic penaltythan does the reaction in solution suggeststhat the ribosome may abrogate the significantsolvent reorganization penalty associated withthe nonenzymatic reaction. Supporting thispossibility, the induced-fit conformationalchange upon binding of the aminoacyl tRNAto the A site appears to organize the PTC struc-turally and electrostatically for catalysis. In thiscontext, the 2′ OH-mediated proton shuttlemechanism provides an appealing and elegantstrategy to facilitate the reaction without the

need for solvent reorganization. However,defining how protons move and how boundwater molecules impart functional capacityremain among the most difficult challenges inenzymology today. As such, the model remainsspeculative despite support from structural,functional, and computational data.

Despite the significant advances, much ofthe current mechanistic understanding comesfrom the analysis of a limited set of P-siteand A-site substrates or substrate analogs. Wehave little knowledge of how the identity ofA-site aminoacyl tRNA or the identity ofthe nascent peptide extending from the P-sitetRNA into the exit tunnel influences peptidyl-transferase kinetics. Additionally, recent studieshave shown that bound antibiotics in combina-tion with specific nascent peptides can lead toprogrammed ribosome stalling. In the future,it will be important to elucidate the structuraland mechanistic basis of how the PTC com-municates other parts of the ribosome, includ-ing the exit tunnel and the locations of boundantibiotics.

SUMMARY POINTS

1. Nitrogen nucleophiles react with carboxylate esters to form amide bonds. The reactioninvolves formation of a tetrahedral intermediate. Following loss of the proton from thenucleophilic nitrogen, the intermediate collapses to form the amide. Ribosomal peptidebond formation likely adheres to this pathway, but the timing of proton transfer stepsand the active-site components that mediate them remain unknown.

2. Ribosome crystallography has revealed a peptidyltransferase center composed of RNArather than protein; however, with the exception of a single 2′-OH group on the P-site peptidyl tRNA, ribosomal RNA does not appear to engage directly in catalysis (seesummary point 5).

3. The 23S RNA active-site residues adopt different conformations depending on the oc-cupancy of the A-site and the type of substrate bound there. In the absence of an A-sitesubstrate, the ribosomal residues sterically protect the P-site bound acyl donor (peptidyl-tRNA) from premature hydrolysis. Accommodation of aminoacyl tRNA in the A siteinduces a conformational change that preorganizes the active site for transfer of thepeptidyl group to the amine nucleophile.

4. Compared to the uncatalyzed reaction, ribosome-catalyzed peptide bond forma-tion occurs with an unfavorable enthalpy of activation and a favorable entropyof activation. The ribosome lowers the activation entropy (T�S‡) of the peptydyl

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transfer reaction by 14 kcal/mol. Insights derived from these observations and compu-tation suggest that, in addition to substrate positioning, preorganization of the activesite ameliorates the solvent reorganization penalty that accompanies the uncatalyzedreaction.

5. The A76 2′ OH of the peptidyl tRNA makes an essential contribution to the peptidyl-transferase reaction. The 2′ OH may participate in a proton shuttle mechanism thattransfers a proton, either directly or indirectly through one or more active-site watermolecules, from the amine nucleophile to either the carbonyl oxygen or the leavinggroup oxygen.

6. During translation termination and as a proofreading mechanism, the peptidyltransferasecenter catalyzes hydrolytic release of the polypeptide from the P-site bound peptidyl-tRNA. In this reaction, binding of release factors to the A site induces conformationalchanges similar to those for peptidyl transfer during translation elongation.

7. Release factors contain a universally conserved GGQmethyl motif that engages in a networkof active-site hydrogen bonds and aids in correct positioning of the attacking waternucleophile.

FUTURE ISSUES

1. Current proposed peptidyl transfer and peptide release mechanisms are based mainlyupon crystallographic and simulation data. The induced-fit mechanism of activationappears well established, but beyond substrate positioning, the role of the ribosome incatalysis remains unclear.

2. Major uncertainty exists regarding several aspects of the mechanism: (a) the timing ofbonding changes along the reaction coordinate, including the proton transfer steps; (b) themolecular and structural basis by which the proton transfer steps are accomplished; and(c) the role of the A76 2′ OH and active-site water molecules in catalysis. Understandingthe precise role of the 2′ OH may illuminate features of the proposed proton shuttlemechanism.

3. Most mechanistic studies have focused on prokaryotic ribosomes. Elucidating the sim-ilarities and differences between prokaryotic ribosomes and eukaryotic ribosomes, bothcytoplasmic and mitochondrial, promises to provide new insights and deepen our under-standing of peptidyltransferase catalysis.

4. The tremendous strides in elucidating the structural and mechanistic basis of chemicalcatalysis by the ribosome have set the stage for identifying functional linkages betweenthe peptidyltransferase center and other aspects of ribosome biology, such as binding ofantibiotics or signal recognition particle, and protein translocation, to name just a few.

5. Fundamental knowledge of the ribosome’s catalytic mechanism may help meet an im-portant future challenge: harnessing the ribosome’s biosynthetic capability to constructdesigner proteins and peptides for use in biomedicine and materials science. Significantadvances on this front have already been made, but more will be needed to exploit thesynthetic potential of this remarkable machine.


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DISCLOSURE STATEMENT

The authors are not aware of any affiliations, memberships, funding, or financial holdings thatmight be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS

We wish to thank all who have contributed in the study of ribosmes. We also thank theNature Publishing Group for permission to use Figure 4. This work was supported in partby the Howard Hughes Medical Institute, National Institutes of Health (NIH) training grantsin Molecular and Cellular Biology, the Chemistry and Biology Interface (5T32GM008720), andNIH grants 1R56AI081987 and 1R01AI081987-01 to J.A.P.

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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

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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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the mechanism of peptidyl transfer catalysis by the ribosome

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