Mechanism of peptide bond formation on the ribosome

Quarterly Reviews of Biophysics 39, 3 (2006), pp. 203–225. f 2006 Cambridge University Press doi:10.1017/S003358350600429X Printed in the United Kingdom First published online 8 August 2006

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Mechanism of peptide bond formation on the ribosome Marina V. Rodnina1*, Malte Beringer1 and Wolfgang Wintermeyer2 1 2

Institute of Physical Biochemistry, University of Witten/Herdecke, Witten, Germany Institute of Molecular Biology, University of Witten/Herdecke, Witten, Germany

Abstract. Peptide bond formation is the fundamental reaction of ribosomal protein synthesis. The ribosome’s active site – the peptidyl transferase center – is composed of rRNA, and thus the ribosome is the largest known RNA catalyst. The ribosome accelerates peptide bond formation by 107-fold relative to the uncatalyzed reaction. Recent progress of structural, biochemical and computational approaches has provided a fairly detailed picture of the catalytic mechanisms employed by the ribosome. Energetically, catalysis is entirely entropic, indicating an important role of solvent reorganization, substrate positioning, and/or orientation of the reacting groups within the active site. The ribosome provides a pre-organized network of electrostatic interactions that stabilize the transition state and facilitate proton shuttling involving ribose hydroxyl groups of tRNA. The catalytic mechanism employed by the ribosome suggests how ancient RNA-world enzymes may have functioned. 1. The ribosome

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2. Peptide bond formation is catalyzed by RNA 205 3. Characteristics of the uncatalyzed reaction

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4. Potential catalytic strategies of the ribosome

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5. Experimental systems 208 6. Substrate binding in the PT center 7. Induced fit in the active site

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8. pH dependence of peptide bond formation 9. Reaction with full-length aa-tRNA 10. Role of active-site residues

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11. pH-dependent structural changes of the active site

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12. Entropic catalysis 217 13. Role of 2¢-OH of A76 in P-site tRNA 218 * Address for correspondence : Dr M. V. Rodnina, Institute of Physical Biochemistry, University of Witten/Herdecke, Stockumer Str. 10, 58448 Witten, Germany. Tel. : +49 2302 926205 ; Fax: +49 2302 926117 ; E-mail: [email protected]

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M. V. Rodnina, M. Beringer and W. Wintermeyer

14. Catalysis by proton shuttling 15. Plasticity of the active site 16. Conclusions

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17. Acknowledgments 18. References

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1. The ribosome The fundamental enzymatic reaction in the biosynthesis of proteins is the formation of the peptide bond. Protein synthesis takes place on the ribosome, a large ribonucleoprotein particle (25 MDa in bacteria) that is made up of three molecules of ribosomal RNA (rRNA) and more than 50 proteins. Functionally, ribosomes are template-dependent polymerases. The ribosome consists of two subunits, the small subunit, 30S, and the large subunit, 50S. The peptidyl transferase (PT) center is located on the 50S subunit of the ribosome (Fig. 1). The substrates of reaction, peptidyl-tRNA (pept-tRNA) and aminoacyl-tRNA (aa-tRNA), are bound to the P and A sites of the ribosome respectively. aa-tRNA is delivered to the A site of the ribosome in a ternary complex with an elongation factor (EF-Tu in bacteria) and GTP according to the match between the tRNA anticodon and the mRNA codon displayed in the decoding site on the 30S subunit. During peptide bond formation, the peptide is transferred from the pept-tRNA to aa-tRNA, yielding deacylated tRNA in the P site and pept-tRNA that is extended by one amino-acid residue in the A site. The subsequent movement of tRNAs and mRNA through the ribosome (translocation) is catalyzed by another elongation factor, EF-G in bacteria, displacing the pepttRNA to the P site and exposing a new codon in the A site for the interaction with the next aa-tRNA. To be compatible with the overall rate of protein synthesis in the cell, the rate of peptide bond formation has to be >10 sx1, and the maximum estimated rate is >300 sx1 (see below). Compared to the reaction in solution, the ribosome accelerates peptide bond formation y107-fold. In recent years, new insights into the mechanism of peptide bond formation were obtained due to the enormous progress in structural, biochemical, and genetic studies of the ribosome. Several atomic-resolution structures of the 50S subunit from an archeon, Haloarcula marismortui, were reported by the Steitz group from Yale, including structures with substrate analogs, several putative transition-state analogs, or products of the reaction bound to the A site and P site (Ban et al. 2000 ; Nissen et al. 2000 ; Hansen et al. 2002 ; Schmeing et al. 2002, 2005a, b). The structures of the 50S subunit from the bacterium Deinococcus radiodurans were solved by the Yonath group at the Weizmann Institute in collaboration with the Max Planck Institute in Berlin (Harms et al. 2001 ; Schlunzen et al. 2001 ; Bashan et al. 2003). The 50S crystal structures led to new ideas regarding the catalytic mechanism of peptide bond formation. This prompted several groups to test these predictions by a number of functional approaches, including ribosome mutagenesis, rapid kinetic studies, and molecular dynamics (MD) simulations. The advances in ribosome genetics allowed the introduction of mutations in rRNA and isolate ribosome mutants which have lethal phenotypes in vivo. Biochemical and kinetic studies characterized the reaction in detail and evaluated the effects of rRNA mutations and substrate modifications. The goal of this review

Mechanism of peptide bond formation on the ribosome

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Fig. 1. Crystal structure of the 50S subunit from H. marismortui in the complex with a transition-state analog (PDB entry 1VQP ; Schmeing et al. 2005a). Ribosomal proteins are blue, the 23S rRNA backbone is brown, and the 5S rRNA backbone olive. Bases are pale green and the transition-state analog red. The figure was generated with Pymol (http://www.pymol.org).

is to summarize these recent findings and present a current model of peptide bond formation on the ribosome. Much of the earlier work can be now incorporated into the framework of the current models ; detailed discussions of the earlier experiments can be found in previous reviews (Krayevsky & Kukhanova, 1979 ; Lieberman & Dahlberg, 1995 ; Barta & Halama, 1996 ; Garrett & Rodriguez-Fonseca, 1996).

2. Peptide bond formation is catalyzed by RNA 50S subunits are composed of two RNA molecules, 23S rRNA and 5S rRNA, and more than 30 proteins. Based on crosslinking studies, the PT center could be located to domain V of 23S rRNA with its interacting proteins (Noller, 1991). Biochemical studies by Noller and colleagues showed that 50S subunits largely depleted of proteins retained PT activity (Noller et al. 1992). The high-resolution crystal structures of the 50S subunit from H. marismortui have revealed that ˚ of the active site (Fig. 2), the PT center is composed of RNA only, with no protein within 15 A ˚ while several proteins, L2, L3, L4, are located within 20–30 A from the catalytic center (Ban et al. 2000). A similar structure was found in 50S subunits from D. radiodurans (Harms et al. 2001 ; Bashan et al. 2003) and in 70S ribosomes from Escherichia coli (Schuwirth et al. 2005). This suggested that the activity might reside in 23S rRNA, although a contribution by residual proteins could not be completely excluded. Previously, evolutionary arguments favored L2 as a candidate for an active-site component of the PT center, since it is one of the most highly conserved proteins of the 50S subunit (Muller & Wittmann-Liebold, 1997). Mutation of the

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Fig. 2. A close view of the transition-state analog (PDB entry 1VQP ; Schmeing et al. 2005a) in the PT center. The P-site-bound CCA part of the analog is orange, the A-site CCA is yellow. The nucleophilic nitrogen is dark blue, and the electrophilic carbonyl carbon green. Ribosomal residues within 15 A˚ of the nucleophile are pale green. The surface of 23S rRNA is brown and surfaces of ribosomal proteins are blue.

conserved His229 in E. coli L2 to Glu led to 50S particles devoid of PT activity (Cooperman et al. 1995). However, other replacements of His229 had only modest effects on the activity of the mutant ribosomes in the synthesis of polyphenylalanine, suggesting that a direct involvement of His229 of L2 in the PT reaction was unlikely (Diedrich et al. 2000). Protein L27 is located close enough to the PT center to interact with the P-site tRNA. The protein was shown to influence the PT activity in E. coli 50S (Bischof et al. 1995). A mutant E. coli strain lacking L27 was viable, but grew 5–6 times more slowly than the wild type and showed deficiencies in the PT activity and impaired binding of tRNA to the A site (Wower et al. 1998). Examination of strains expressing L27 variants truncated at the N terminus revealed that the absence of as few as three amino acids leads to a decrease in growth rate, an impairment in PT activity, and a sharp decline in the labeling of L27 from the 3k-end of a photoreactive tRNA at the ribosomal P site (Maguire et al. 2005). These findings may suggest that the flexible N-terminal sequence of L27, which protrudes onto the interface of the bacterial 50S subunit, can reach the PT active site and contribute to its function. However, H. marismortui ribosomes do not have protein L27 or any homologous counterpart, indicating that L27 cannot be part of an evolutionary conserved PT mechanism which is expected to employ the same residues in all organisms. Furthermore, in the crystal structure of E. coli ribosomes the N-terminus of L27 is located too far away from the PT center to be directly involved in catalysis (Schuwirth et al. 2005). These data suggest that L27 may contribute to peptide bond formation indirectly by helping to correctly position tRNA substrates at the catalytic site (Maguire et al. 2005), rather than by taking part in catalysis directly.

Mechanism of peptide bond formation on the ribosome O

O O + R2

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−

R3 R1

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k1 k−1

NH2

Step 1

O R1 H

+

O

N H R2

T±

R3

k2[B] k−2[BH]

R2

O R1 H

Step 2

O N

T−

R2

R3

k3[BH] k−3[B]

R1

N H +

R3 OH

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Fig. 3. Scheme of the aminolysis reaction mechanism. Step 1, formation of the zwitterionic tetrahedral intermediate T¡. Step 2, deprotonation of the positively charged amino nitrogen resulting in the second intermediate, Tx. Step 3, product formation. Proton transfers during the deprotonation of T¡ (step 2) and the formation the leaving group (step 3) can be catalyzed by base and acid respectively. R1, R2, R3, substituents.

3. Characteristics of the uncatalyzed reaction Uncatalyzed peptide bond formation was studied using different model substrates. When amino acids and aminoacyl-adenylates were used as attacking nucleophile and reactive ester respectively, reaction rates were very low, about 10x5 Mx1 sx1, and varied 20-fold, depending on the identity of the amino acid, which probably reflected the differences in the pKa values of the attacking a-NH2 group (Weber & Orgel, 1979). Non-enzymatic ester aminolysis reactions are predicted to proceed through two tetrahedral intermediates (Fig. 3). Early studies by Jencks and co-workers showed that the aminolysis of methyl formate by different amine nucleophiles proceeds via general base-catalyzed attack by the unprotonated amine at high pH (Satterthwait & Jencks, 1974 ; Blackburn & Jencks, 1986). As the reaction pH is lowered, there is a break in the pH profile, suggesting a change in the ratedetermining step of the reaction. At high pH, the attack on the ester carbonyl carbon by the amino nitrogen (step 1) is rapid and reversible, and trapping of the tetrahedral intermediate T¡ by general base-catalyzed proton removal is rate-determining (step 2). At low pH, the breakdown of the tetrahedral intermediate Tx becomes rate-determining (step 3) (Satterthwait & Jencks, 1974). A late rate-limiting step is reflected in a high Brønsted coefficient, which is 08 (Jencks & Gilchrist, 1968). The reaction between the primary amine Tris(hydroxymethyl)aminomethane and organic esters such as N-formylglycine ethylene glycol ester (10x4 Mx1 sx1) showed a large unfavorable activation entropy (TDS$=–131 kcal molx1) and enthalpy (DH$=91 kcal molx1) (Sievers et al. 2004). Aminolysis of a natural substrate of the PT reaction, fMet-tRNAfMet, had a more favorable entropic term (TDS$=x65 kcal molx1), and a somewhat less favorable activation enthalpy (DH$=162 kcal molx1) (Rodnina et al. 2005). Recent theoretical work by Warshel and colleagues (Sharma et al. 2005) suggested that a significant part of the observed activation entropy of the solution reaction is due to the entropy of solvent reorganization.

4. Potential catalytic strategies of the ribosome Enzymes employ a variety of strategies to increase reaction rates, including proper positioning of substrates, stabilization of the transition state, destabilization of the ground state, and chemical catalysis, such as general acid-base catalysis. All these strategies might be used by the ribosome. Charged intermediates and/or transition states develop during the PT reaction, and the ribosome might catalyze the reaction by stabilizing them by electrostatic interactions or

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abstracting/donating of protons. The T¡ intermediate of the PT reaction contains a positively charged amino nitrogen and an oxyanion linked to the tetrahedral carbon which would be stabilized by electrostatic interactions with the ribosome. After the nucleophilic attack, a proton from the positively charged amino nitrogen may be abstracted by a putative general base to generate an uncharged secondary amine. A proton is required to form the leaving group, the 3kOH of the deacylated tRNA in the P site, which may require a general acid. For general acid-base catalysis to occur, the pKa values of the catalytic groups have to be close to neutrality in order to efficiently abstract or donate a proton during the reaction. The unperturbed pKa values of RNA bases are far from neutrality, i.e. 35 and 42 for A and C and 92 for G and U respectively (Bevilacqua et al. 2004). Thus, if rRNA bases were to take part in chemical catalysis in an aqueous environment at neutral pH, their pKa values would have to be shifted quite significantly by a particular chemical environment. In addition, the efficiency of acid-base catalysis depends on Brønsted coefficients. Ionizing groups with pKa values outside the neutral pH may be efficient acid-base catalysts only when their Brønsted coefficients are sufficiently high. Thus, if acid-base catalysis contributed significantly to peptide bond formation, then the reaction rate should depend on the ionization of residues acting as general acid-base catalysts, and the Brønsted coefficient should be high. Furthermore, enzymes that employ general acid-base or covalent catalysis act by lowering the activation enthalpy of the reactions they catalyze. The entropic contribution for the formation of the transition state in those cases is small and variable, with an average value close to zero (Wolfenden et al. 1999). Finally, the ribosome may promote peptide bond formation by a mechanism that differs in detail from the uncatalyzed aminolysis reaction in solution, as suggested by the recent measurements of kinetic isotope effects for the PT reaction on isolated 50S subunits (Seila et al. 2005). To understand the mechanism of catalysis by the ribosome, several important questions have to be solved. The reaction pathways of the catalyzed and uncatalyzed reactions have to be compared and the rate-limiting steps have to be identified. The structure of the active site has to be known. The participation of groups potentially involved in the reaction has to be examined. Ionizing groups contributing to catalysis have to be identified. Finally, the activation parameters of the catalyzed and uncatalyzed reactions have to be measured and compared. These issues can only be resolved by applying a combination of structural studies, kinetics, ribosome mutagenesis, and chemical replacement of the reacting groups. In the following, we will focus on the results of these studies.

5. Experimental systems In the cell, peptide bond formation takes place on 70S ribosomes where two tRNAs, pept-tRNA and aa-tRNA are bound to their respective mRNA codons at the P and A sites. Clearly, it would be desirable to investigate the mechanism of peptide bond formation in this system, which is the biologically relevant one. However, high-resolution structural information so far is available only for isolated 50S subunits. Although the crystal structures of 70S ribosomes (Yusupov et al. 2001 ; Schuwirth et al. 2005) provide a wealth of useful information and allow for a better interpretation of the biochemical and genetic data, the resolution of the structure of ribosome–tRNA complexes is insufficient to see details of the interactions at the PT center. An additional problem is that the crystal structures of E. coli ribosomes, for which most of the biochemical and kinetic information is available, were obtained for vacant ribosomes without tRNA ligands.


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Fig. 4. Experimental approach to study the kinetics of peptide bond formation. Upper panel, reaction substrates, pept-tRNA (left) and Pmn (right). Lower panel, scheme of the experiment. The ribosomemRNApept-tRNA complex is rapidly mixed with Pmn in a quench-flow apparatus. The reaction is stopped by the addition of quencher (e.g. KOH or formic acid) and the reaction product, pept-Pmn, is analyzed by HPLC.

Another important issue that has to be addressed, particularly when mutational and biochemical studies are concerned, is the identification of the rate-limiting step of the reaction. In the complete translation system, the accommodation of the 3k-end of aa-tRNA in the PT center is rate-limiting for the following peptide bond formation, while the peptidyl transfer is intrinsically rapid (Pape et al. 1998). Therefore, the properties of the PT reaction itself cannot be studied with unmodified, full-length aa-tRNA. Furthermore, the overall rate of protein synthesis, 10–20 sx1, is too fast to be followed kinetically by conventional biochemical assays. The latter limitation was overcome by introducing the quench-flow technique for studying the PT reaction (Katunin et al. 2002) (Fig. 4). In these experiments, ribosomes programmed with natural mRNA and carrying fMet-tRNA or pept-tRNA in the P site were reacted with the antibiotic puromycin (Pmn ; O-methyl tyrosine linked to N6-dimethyl adenosine via an amide bond) as A-site substrate analog. The reaction rate was not limited by Pmn binding (Sievers et al. 2004) and the kinetics of the catalytic step could be monitored ; the maximum rate of peptide bond formation, measured with pept-tRNA as P-site substrate, was y50 sx1 at pH>8 (Katunin et al. 2002 ; Youngman et al.

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2004). The rate of reaction varied within a factor of 50–100 depending on length of the peptidyl moiety of the P-site tRNA and/or different C-terminal amino acid of the peptide : the reaction rate was close to 1 sx1 with fMet-tRNAfMet in the P site, 10–20 sx1 with di- and tri-peptidyltRNA (Katunin et al. 2002 ; Brunelle et al. 2006), and 50 sx1 with fMetAlaAsnMetPheAla-tRNA (Katunin et al. 2002). The mechanism by which a longer peptide chain on pept-tRNA brings about the increase of the reaction rate is unclear. Early functional assays utilized the so-called fragment reaction containing isolated 50S subunits and oligonucleotide fragments of the 3k-terminus of tRNA, typically CACCA, CCA and CA charged with fMet, AcLeu, or AcPhe, or even N-blocked amino-acid derivatives linked to adenosine as P-site substrates, and Pmn (Monro, 1967 ; Monro & Marcker, 1967 ; Pestka, 1972 ; Krayevsky & Kukhanova, 1979 ; Dorner et al. 2003). Reactions between small substrate analogs on isolated 50S subunits were slower by several orders of magnitude, compared to 70S ribosomes. The large rate differences could be due to using non-saturating concentrations of substrates or else could imply principal – mechanistic and structural – differences between the reactions on 70S ribosomes and isolated 50S subunits. However, when full-size fMet-tRNAfMet and Pmn or C-Pmn were used as peptide donor and acceptor substrates respectively, the reaction on 50S subunits and 70S ribosomes proceeded at the same rate, indicating that the intrinsic activity of 50S subunits is not different from that of 70S ribosomes ( Wohlgemuth et al. 2006). The reactions catalyzed by the 70S ribosome and the 50S subunit are similar also in a number of other ways : they are susceptible to the same inhibitors (Moore & Steitz, 2003 ; Poehlsgaard & Douthwaite, 2005 ; Polacek & Mankin, 2005 ; Yonath, 2005), and have similar pH profiles (Maden & Monro, 1968 ; Katunin et al. 2002 ; Okuda et al. 2005 ; Brunelle et al. 2006). This argues that functional assays performed on 70S ribosomes may be interpreted on the basis of the high-resolution crystal structures of 50S subunits. 6. Substrate binding in the PT center The acceptor arms of the A- and P-site tRNAs are located in a cleft of the 50S interface side (Yusupov et al. 2001 ; Hansen et al. 2002). Their universally conserved CCA ends are oriented and held in place by interactions with 23S rRNA (Fig. 5 a). In the P site, C74 and C75 of the tRNA are base-paired to G2251 and G2252 of the P loop of 23S rRNA (Samaha et al. 1995 ; Nissen et al. 2000 ; Schmeing et al. 2005a). (Note that here and following we shall use the rRNA numbering of E. coli ribosomes.) The CCA end of the A-site tRNA is fixed by base-pairing of C75 with G2553 of the A loop of 23S rRNA (Kim & Green, 1999 ; Nissen et al. 2000 ; Schmeing et al. 2005a). The 3k-terminal A76 of both A- and P-site tRNAs form interactions with residue G2583 and base pair A2450-C2501 respectively. The conserved bases A2451, U2506, U2585, C2452 and A2602 are located in the core of the PT center (Bashan et al. 2003 ; Schmeing et al. 2005a, b) (Fig. 5 b). A somewhat different neighborhood of the reactive a-NH2 group was reported for a substrate analog attached to a short RNA hairpin bound to D. radiodurans 50S subunits (Bashan et al. 2003). The reason for this discrepancy is not clear, and its solution awaits high-resolution structures of subunits with different substrate analogs or, preferably, full-size aminoacyl- and peptidyltRNAs. The crystal structures of H. marismortui 50S subunits complexed with different transitionstate analogs revealed that the reaction proceeds through a tetrahedral intermediate with S chirality. The oxyanion of the tetrahedral intermediate is stabilized by a water molecule that is positioned by nucleotides A2602 and U2584 (Schmeing et al. 2005a). The only atom within

Mechanism of peptide bond formation on the ribosome (a)

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(b)

Fig. 5. Binding of substrates at the PT center of the ribosome. (a) Interactions of the tRNA analogs, CChPmn ( yellow) and CCA-pcb (see text) (orange) with the residues of 23S rRNA (PDB entry 1VQN ; Schmeing et al. 2005b). The attacking nucleophilic amino group (circled red) is blue, and the attacked carbonyl carbon green. Hydrogen bonds are dashed lines. ‘Inner-shell ’ nucleotides are omitted for clarity. (b) Close-up view of rRNA residues (magenta) at the ‘inner shell’ of the PT center. Dashed lines indicate hydrogen bonds from the attacking nucleophile to N3 of A2451 and to 2k-OH of A76 (P-site substrate) respectively, in the pre-reaction induced state (Schmeing et al. 2005b).

hydrogen-bonding distance of the a-NH2 group mimic was the 2k-OH of A76 of the P-site portion of the transition-state analog (Schmeing et al. 2005a). N3 of A2451, which is within hydrogen-bonding distance in pre-reaction states (see below), appears to lose this interaction with the attacking group during the course of the reaction.

7. Induced fit in the active site Recently, Steitz and colleagues solved the structures of a number of 50S complexes with A-site substrate analogs of different lengths (Schmeing et al. 2005b). The smallest A-site substrate contained the terminal C75 and A76 of the tRNA and the aminoacyl moiety. The substrate was bound to the 50S subunit through the typical A site-binding contacts (see above) and the conformation of active site residues in the complex was similar to that with an empty A site. The Psite substrate, CCA-pcb, a CCA oligonucleotide linked to an analog of the growing peptide chain, exhibited a conformation that was unfavorable for the PT reaction, as the carbonyl oxygen was pointing towards the a-NH2 group of the A site-substrate mimic and the atoms that are to react ˚ apart. were y4 A Binding of an A-site substrate that additionally included C74 induced a conformational change in the PT center (Schmeing et al. 2005b). The stacking of the CCA bases positioned the substrate closer to the center of the active site and involved rearrangements of residues G2583, U2506, and U2585. These rearrangements resulted in a conformation of the active site in which the carbonyl carbon of peptidyl-tRNA was more appropriately oriented for the attack by the nucleophile. In this pre-reaction ground state, the reactive a-NH2 group formed hydrogen bonds with both N3 of A2451 and the 2k-OH of A76 of the P-site substrate (Fig. 5 b). The conformation of the rRNA in this complex was nearly identical to that observed in the complex with transition-state analogs, suggesting that rRNA rearrangements induced by substrate binding are relevant to the PT reaction pathway. These structures suggest an induced-fit mechanism in which binding of the correct substrate to the A site induces repositioning of both substrates and activesite residues to promote the PT reaction (Schmeing et al. 2005b).

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One implication of the induced-fit model is that the rate of peptide bond formation with CCPmn as A-site substrate should be higher than with C-Pmn. However, measurements at conditions of substrate limitation did not detect gross changes in the rates of catalysis (Brunelle et al. 2006). Surprisingly, at conditions of substrate saturation the rate of peptide bond formation with CC-Pmn, 4 sx1, was even lower than that with C-Pmn, 13 sx1 (M. Beringer, unpublished observations). MD simulations suggested that the U2585 base is fairly mobile and spans a range of ˚ qvist, 2006). conformations between the two steps observed in the crystal structures (Trobro & A Also the activation energies of the induced and uninduced reactions were found to be quite similar (Trobro & A˚qvist, 2006), in agreement with the biochemical data. These results indicated that the effect of induced fit on the rate of peptidyl transfer is small. Additionally, the reaction rate with CC-Pmn was independent of pH (M. Beringer, unpublished observations), whereas the rate/pH profile of the reaction with C-Pmn shows a single pKa close to 7, most probably reflecting the ionization of the a-NH2 group of C-Pmn (Brunelle et al. 2006). These results suggest that, depending of the substrate in the A site, the details of the reaction pathway may change and different elemental steps may determine the reaction rate. While the chemistry step determines the rate of the reaction with Pmn (and possibly C-Pmn) (Katunin et al. 2002 ; Sievers et al. 2004), the rate of PT reaction with the full-size aa-tRNA is limited by the preceding step of aa-tRNA accommodation in the A site (Pape et al. 1998 ; Bieling et al. 2006). The latter step is independent of pH and, because it is rate-limiting, masks the ionization of groups involved in peptidyl transfer, i.e. the aNH2 group of amino acid [ pKa=8 ( Wolfenden, 1963)]. Likewise, binding of CC-Pmn to the ribosome (or the rearrangements at the active site) may be sufficiently slow to limit the rate of the following steps ; hence the reduced rate and lack of pH dependence of reaction. This suggests that interactions of C74 with the ribosome change the reaction pathway and, therefore, the effect of induced fit on the chemistry step of peptide bond formation cannot be assessed biochemically. Binding of the correct P-site substrate, the full-length pept-tRNA, results in a much faster PT reaction, compared to short substrate analogs ( Wohlgemuth et al. 2006). The rapid reaction of fMet-tRNAfMet on 50S subunits, compared to slowly reacting oligonucleotide substrate analogs, suggests that full-size tRNA in the P site may be important for maintaining the active conformation of the PT center ( Wohlgemuth et al. 2006). In fact, the reaction on 50S subunits was faster when full-size Ala-tRNAAla, rather than a short 3k-terminal fragment, was used as P-site substrate (Sardesai et al. 1999). This raises the possibility that an interaction of a part of the tRNA molecule beyond the 3k-terminal sequence induces a conformational change of the PT center, thus enhancing the catalytic activity. Except for the contacts in the PT center itself, tRNA interactions with the 50S subunit involve elements (helix 69 of 23S rRNA ; protein L5) that contact the elbow region of the P-site tRNA (Yusupov et al. 2001). These contacts may be present in the complexes of the 50S subunit with fMet-tRNAfMet, but not in those with oligonucleotide substrate analogs. The contact site is probably located in the acceptor domain of the tRNA, because Ala-tRNAAla and an Ala-tRNA mini-helix were equally active on 50S subunits, whereas a 9-mer 3k-terminal fragment of the same tRNA had a much lower activity (Sardesai et al. 1999). The only contact of the acceptor domain with the 50S subunit is through protein L5 which, therefore, may be involved in modulating the PT activity. 8. pH dependence of peptide bond formation Early studies revealed that the PT reaction is strongly pH-dependent (Maden & Monro, 1968 ; Pestka, 1972). The shortcoming of the early experiments was that reaction rates were quite low,


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3 2

log(kpep)

1 0 –1 –2 –3 –4 5

6

7 pH

8

Fig. 6. pH dependence of catalysis. Reactions between fMetPhe-tRNAPhe and Pmn ($), fMet-tRNAfMet and Phe-tRNAPhe (#), or fMet-tRNAfMet and hydroxy-Phe-tRNAPhe (m). The dashed gray line indicates a modeled pH dependence of peptide bond formation with Phe-tRNAPhe calculated assuming a single ionizing group (pKa1=80 for the a-NH2 group of aa-tRNA) and kpep=5 sx1 at pH=65.

in the minutes range, suggesting that the observed rates were limited by step(s) other than the chemical step. Rapid kinetic measurements performed with fMetPhe-tRNAPhe as P-site substrate and Pmn as A-site substrate, in which the rate of the chemistry step was monitored, demonstrated that the reaction rate, in fact, was strongly pH-dependent (Katunin et al. 2002) (Fig. 6). The effect was not due to ribosome inactivation (Bayfield et al. 2001), because the extent of the reaction remained close to 100 % over the whole pH range studied. In addition to the inhibition by protonation of the nucleophilic a-NH2 group [ pKa=69 in Pmn (Katunin et al. 2002)], the reaction was also inhibited by protonation of a group of the ribosome with a pKa around 75. When hydroxy-Pmn (h-Pmn), a puromycin derivative where the a-NH2 group was replaced by an OH group, was used as A-site substrate, the reaction was slower, because of the decreased nucleophilicity of the attacking group. Furthermore, the reaction rate became dependent only on one ionizing group with a pKa=75, and because h-Pmn does not possess any ionizing group within the pH range studied, this group must reside on the ribosomal complex. The data revealed that upon protonation of this ribosomal group, and, with Pmn in the deprotonated state, the reaction still proceeded five orders of magnitude faster than the uncatalyzed reaction. This pHinsensitive part of the overall catalysis may be attributed to substrate positioning, proximity effects, electrostatic stabilization of the transition state, or desolvation. The second conclusion is that the pH-sensitive part of the reaction, which is due to protonation of a ribosomal group with pKa=75, contributes another factor of 100 to the overall rate. While this could be due to general acid-base catalysis, protonation could equally well induce a conformation of the active site in which the approach to the transition state is impaired for structural reasons. In the latter case,

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which seems likely (see below), mainly substrate positioning, electrostatics, and related effects would be responsible for catalysis. 9. Reaction with full-length aa-tRNA The rate of peptide bond formation with full-length aa-tRNA is limited by the preceding accommodation step ; hence direct measurements of the pH dependence of the chemistry step are not feasible with aa-tRNA. If an ionizing group on the ribosome with pKa in the neutral range were involved in the reaction, and given that the pKa of the a-NH2 group of aa-tRNA is around 80 (Wolfenden, 1963), then the reaction rate between fMet-tRNAfMet in the P site and PhetRNAPhe in the A site should strongly decrease when the pH is lowered below neutrality. Eventually, peptide bond formation could become slower than accommodation, which is independent of pH in the range between 6 and 9, and it could be possible to study the chemistry step in the range below the critical pH. However, the apparent rate of peptide bond formation remained at 5 sx1, which equals the rate of aa-tRNA accommodation, at pH values down to 60 (Bieling et al. 2006). This result may be explained in two ways : either the rate of the PT reaction is not affected by ionization of ribosomal residues or, alternatively, the chemical step intrinsically is so rapid that the protonation of a ribosomal group contributing to catalysis does not inhibit peptide bond formation to the extent that it becomes slower than accommodation. In order to resolve the issue experimentally, we have resorted to lowering the reactivity of the A-site substrate such that accommodation was no longer rate-limiting for the reaction (Bieling et al. 2006). This was accomplished by replacing the a-NH2 group in Phe-tRNA by an OH group, in analogy to the hydroxy-Pmn derivative described above. The hydroxyl derivative of PhetRNA was fully reactive with fMet-tRNA in the P site, but with a much lower rate of 10x3 sx1, i.e. far below the rate of accommodation. Measuring the reaction rate at different pH values in the range between 6 and 9 revealed that the reaction rate was not affected by pH changes (Fig. 6). This result indicates (i) that catalysis by the PT center intrinsically is independent of pH and (ii) that full-length (hydroxy) aa-tRNA stabilizes the active conformation of the PT center to such an extent that it becomes insensitive to pH. This conclusion is corroborated by results obtained with C-Pmn as A-site substrate : the reaction between C-Pmn and P-site fMetPhe-tRNA was dependent on ionization of the a-NH2 group of Pmn, but not of ribosomal groups, suggesting that the presence of C75, and presumably its interaction with G2553, was sufficient to induce and stabilize the active conformation of the PT center (Brunelle et al. 2006). These data argue against an involvement of ionizing groups of the ribosome in the catalytic mechanism of the PT reaction, and indicate that the ribosome does not utilize general acid-base catalysis to any significant extent. The rate of peptide bond formation with unmodified aa-tRNA is estimated as >300 sx1 (Fig. 6), assuming pKa=8 for the a-NH2 group as the only ionizing group affecting the reaction (Bieling et al. 2006). The fact that no pH dependence was observed between pH 6 and 9 does not by itself eliminate acid-base catalysis, because a combination of an acid and a base with pKa values outside this range may result in the same behavior. In such a case, the pH independence is not due to the lack of ionization events, but instead is due to the decrease in the level of the functional form of the acid being offset by the increase in the functional form of the base (Bevilacqua, 2003). There are examples of ribozymes using acid-base catalysis by nucleobases with pKa values well outside the neutral range (Bevilacqua et al. 2004 ; Fedor & Williamson, 2005). Notably, the reaction rates attained by such catalysts are moderate, usually 300 sx1, the rate of peptide bond formation on those ribosomes which have their general base and acid in their respective ionization states required for the reaction would have to proceed at >3107 sx1, i.e. unrealistically fast, whereas the rate in other ribozymes would not have to be larger than y103 sx1. Furthermore, kmax itself is a function of the Brønsted coefficient. In most ribozymes, the Brønsted coefficient is y05 (Bevilacqua, 2003), but appears to be very low,