Synthetic peptide model of an essential region of an aminoacyl-tRNA synthetase

A 40 amino acid sequence of the unsolved structure of Escherichia coli alanine-tRNA synthetase is essential for tRNA binding and encodes an immunological determinant that cross-reacts with antibodies raised against a eukaryote (insect Bombyx mori) alanine enzyme. The secondary structure of this sequence is predicted to be an amphiphilic alpha-helix that includes one aspartyl and eight glutamyl side chain carboxyl groups. The antibody reactivity and the conformation of a synthetic peptide model of this region (Glu346 to Ser385) were investigated. In addition, double Arg----Gln and Leu----Ala substitutions were separately placed in the enzyme on the hydrophilic and hydrophobic face, respectively, of the predicted helix. These mutations conserve the polar/nonpolar character of each face and retain the potential for helix formation. Circular dichroism spectra of the synthetic peptide model demonstrate the potential for amphiphilic helix formation for the segment from Glu346 to Ser385. The behavior of the mutations in the enzyme, together with earlier data and immunological assays presented here, suggests that one face of the putative helix is an antigenic region of the surface of the enzyme where it contributes to the interaction with alanine tRNA and that the specific sequence of the helix is an important determinant of enzyme stability.     

Gamma-glutamylcysteine synthetase. Interactions of an essential sulfhydryl group

gamma-Glutamylcysteine synthetase (isolated from rat kidney) has one sulfhydryl group that reacts with 5,5'-dithiobis-(2-nitrobenzoate). This single exposed sulfhydryl group is not required for enzyme activity. The enzyme is potently inactivated by cystamine, which apparently interacts with a sulfhydryl group at the active site to form a mixed disulfide. 5,5'-Dithiobis-(2-nitrobenzoate) does not interact with the sulfhydryl group that reacts with cystamine. After the enzyme was 90% inactivated by reaction with cystamine, 3.4 mol of 5,5'-dithiobis-(2-nitrobenzoate) reacted per mol of enzyme, indicating that binding of cystamine exposes sulfhydryl groups which are apparently buried or unreactive in the native enzyme. L-Glutamate (but not D-glutamate or L-alpha-aminobutyrate) protected against inactivation by cystamine. In contrast, ATP enhanced the rate of inactivation by cystamine, and the apparent Km value for this effect is similar to that for ATP in the catalytic reaction. Studies on the structural features of cystamine that facilitate its interaction with the enzyme showed that selenocystamine, monodansylcystamine, and N-[2[2-aminoethyl)-dithio)ethyl]-4-azido-2-nitrobenzeneamine are also good inhibitors. Whereas S-(S-methyl)cysteamine-Sepharose does not interact with the enzyme (Seelig, G. F., and Meister, A. (1982) J. Biol. Chem. 257, 5092-5096), S-(S-methyl)cysteamine is a potent inhibitor; 1 mol of this compound completely inactivated 1 mol of enzyme. In the course of this work, a useful modification of the method for isolating this enzyme from kidney was developed.     

Catalytic peptide of human glutaminyl-tRNA synthetase is essential for its assembly to the aminoacyl-tRNA synthetase complex

Human glutaminyl-tRNA synthetase (QRS) is one of several mammalian aminoacyl-tRNA synthetases (ARSs) that form a macromolecular protein complex. To understand the mechanism of QRS targeting to the multi-ARS complex, we analyzed both exogenous and endogenous QRSs by immunoprecipitation after overexpression of various Myc-tagged QRS mutants in human embryonic kidney 293 cells. Whereas a deletion mutant containing only the catalytic domain (QRS-C) was targeted to the multi-ARS complex, a mutant QRS containing only the N-terminal appended domain (QRS-N) was not. Deletion mapping showed that the ATP-binding Rossman fold was necessary for targeting of QRS to the multi-ARS complex. Furthermore, exogenous Myc-tagged QRS-C was co-immunoprecipitated with endogenous QRS. Since glutaminylation of tRNA was dramatically increased in cells transfected with the full-length QRS, but not with either QRS-C or QRS-N, both the QRS catalytic domain and the N-terminal appended domain were required for full aminoacylation activity. When QRS-C was overexpressed, arginyl-tRNA synthetase and p43 were released from the multi-ARS complex along with endogenous QRS, suggesting that the N-terminal appendix of QRS is required to keep arginyl-tRNA synthetase and p43 within the complex. Thus, the eukaryote-specific N-terminal appendix of QRS appears to stabilize the association of other components in the multi-ARS complex, whereas the C-terminal catalytic domain is necessary for QRS association with the multi-ARS complex.     

Decoding the impact of disease-causing mutations in an essential aminoacyl-tRNA synthetase

Aminoacyl-tRNA synthetases are housekeeping enzymes that catalyze the specific attachment of amino acids onto cognate tRNAs, providing building blocks for ribosomal protein synthesis. Owing to the absolutely essential nature of these enzymes, the possibility that mutations in their sequence could be the underlying cause of diseases had not been foreseen. However, we are learning of patients bearing familial mutations in aminoacyl-tRNA synthetases at an exponential rate. In a recent issue of JBC, Jin et al. analyzed the impact of two such mutations in the very special bifunctional human glutamyl-prolyl-tRNA synthetase and convincingly decode how these mutations elicit the integrated stress response.     

Cyanylation of 3-hydroxybutyrate dehydrogenase. The "essential" sulfhydryl group is not involved in catalysis

3-Hydroxybutyrate dehydrogenase is a lipid-requiring enzyme with an absolute requirement of lecithin for function. The enzyme contains two sulfhydryl groups per monomer. Modification of the more reactive sulfhydryl group with N-ethylmaleimide resulted in inactivation of the enzyme and modification of coenzyme-binding characteristics [McIntyre, J. O., Fleer, E. A. M. and Fleischer, S. (1984) Biochemistry 23, 5135-5141]. The present study further investigates the function of the sulfhydryl groups by utilizing chemical derivatization techniques. The reactive sulfhydryl was derivatized first with 3,3'-dithiobis(6-nitrobenzoic acid) (Ellman's reagent) to form the S-(carboxynitrophenylthio) derivative which could then be replaced with cyanide to form the S-cyanylated enzyme. We find that derivatizing the essential sulfhydryl group leads to some loss of activity. The effect appears to be steric since a larger derivatizing group gives greater loss of activity. The normal enzyme is inhibited approximately 50% in excess substrate. Derivatization of the reactive sulfhydryl group results in loss of this substrate inhibition, the modified enzyme being at least three-fold more active at high substrate concentrations; the activity increases from 18% to 54% and from 1% to 4% of maximal activity for the S-cyanylated and S-(carboxynitrophenylthio) enzyme derivatives, respectively. Cyanylation results in complete loss of fluorescence energy transfer from tryptophan to NADH at low salt concentration but is normal in the presence of 100mM NaCl. However, the binding constant of the coenzyme is decreased only several-fold in the cyanylated enzyme as studied by fluorescence quenching. The cyanylated enzyme formed tight ternary complexes (spin-labeled NADH-monomethylmalonate) (spin-labeled NAD-sulfite) similar to that formed by the normal enzyme. The spin label is highly immobilized, but the hyperfine splitting values differ somewhat from the normal enzyme. We conclude that the reactive sulfhydryl is close to the active site of 3-hydroxybutyrate dehydrogenase but is not involved in the catalytic mechanism.     

The C2 domain of phosphatidylserine decarboxylase 2 is not required for catalysis but is essential for in vivo function

Phosphatidylserine decarboxylase 2 (Psd2p) is currently being used to study lipid trafficking processes in intact and permeabilized yeast cells. The Psd2p contains a C2 homology domain and a putative Golgi retention/localization (GR) domain. C2 domains play important functions in membrane binding and docking reactions involving phospholipids and proteins. We constructed a C2 domain deletion variant (C2Delta) and a GR deletion variant (GRDelta) of Psd2p and examined their effects on in vivo function and catalysis. Immunoblotting confirmed that the predicted immature and mature forms of Psd2(C2Delta)p, Psd2(GRDelta)p, and wild type Psd2p were produced in vivo and that the proteins localized normally. Enzymology revealed that the Psd2(C2Delta)p and Psd2(GRDelta)p were catalytically active and could readily be expressed at levels 10-fold higher than endogenous Psd2p. Both Psd2p and Psd2(GRDelta)p expression complemented the growth defect of psd1Deltapsd2Delta strains and resulted in normal aminoglycerophospholipid metabolism. In contrast, the Psd2(C2Delta)p failed to complement psd1Deltapsd2Delta strains, and [(3)H]serine labeling revealed a severe defect in the formation of PtdEtn in both intact and permeabilized cells, indicative of disruption of lipid trafficking. These findings identify an essential, non-catalytic function of the C2 domain of Psd2p and raise the possibility that it plays a direct role in membrane docking and/or PtdSer transport to the enzyme.     

The active site sulfhydryl of aconitase is not required for catalytic activity

Previous reports have demonstrated that aconitase has a single reactive sulfhydryl at or near the active site (Johnson, P. G., Waheed, A., Jones, L., Glaid, A. J., and Gawron, O. (1977) Biochem. Biophys. Res. Commun. 74, 384-389). On the basis of experiments with phenacyl bromide in which enzyme activity was abolished while substrate afforded protection, it was concluded that this group was an essential sulfhydryl. We have further examined the reactivity of this group and confirmed the result that, when reagents with bulky groups (e.g. N-ethylmaleimide or phenacyl bromide) modify the protein at the reactive sulfhydryl, activity is lost. However, when smaller groups, e.g. the SCH3 from methylmethanethiosulfonate or the CH2CONH2 from iodoacetamide, are introduced, there is only partial (50%) or no loss of activity. Experiments were performed to obtain evidence that these reagents are modifying the same residue. Methylmethanethio-sulfonate-treated enzyme showed an increase in the Km for citrate from 200 to 330 microM. EPR spectra were taken of the reduced N-ethylmaleimide- and iodoacetamide-modified enzyme in the presence of substrate. The former gave a spectrum typical of the substrate-free enzyme, while the spectrum of the latter was identical to enzyme with bound substrate. We, therefore, conclude that modification of this sulfhydryl affects activity by interfering with the binding of substrate to the active site and is not essential in the catalytic process.     

A new mechanism of post-transfer editing by aminoacyl-tRNA synthetases: catalysis of hydrolytic reaction by bacterial-type prolyl-tRNA synthetase

Aminoacyl tRNA synthetases are enzymes that specifically attach amino acids to cognate tRNAs for use in the ribosomal stage of translation. For many aminoacyl tRNA synthetases, the required level of amino acid specificity is achieved either by specific hydrolysis of misactivated aminoacyl-adenylate intermediate (pre-transfer editing) or by hydrolysis of the mischarged aminoacyl-tRNA (post-transfer editing). To investigate the mechanism of post-transfer editing of alanine by prolyl-tRNA synthetase from the pathogenic bacteria Enterococcus faecalis, we used molecular modeling, molecular dynamic simulations, quantum mechanical (QM) calculations, site-directed mutagenesis of the enzyme, and tRNA modification. The results support a new tRNA-assisted mechanism of hydrolysis of misacylated Ala-tRNA^Pro. The most important functional element of this catalytic mechanism is the 2′-OH group of the terminal adenosine 76 of Ala-tRNA^Pro, which forms an intramolecular hydrogen bond with the carbonyl group of the alanine residue, strongly facilitating hydrolysis. Hydrolysis was shown by QM methods to proceed via a general acid-base catalysis mechanism involving two functionally distinct water molecules. The transition state of the reaction was identified. Amino acid residues of the editing active site participate in the coordination of substrate and both attacking and assisting water molecules, performing the proton transfer to the 3′-O atom of A76.     

A unique insert of leucyl-tRNA synthetase is required for aminoacylation and not amino acid editing

Leucyl-tRNA synthetase (LeuRS) is a class I enzyme, which houses its aminoacylation active site in a canonical core that is defined by a Rossmann nucleotide binding fold. In addition, many LeuRSs bear a unique polypeptide insert comprised of about 50 amino acids located just upstream of the conserved KMSKS sequence. The role of this leucine-specific domain (LS-domain) remains undefined. We hypothesized that this domain may be important for substrate recognition in aminoacylation and/or amino acid editing. We carried out a series of deletion mutations and chimeric swaps within the leucine-specific domain of Escherichia coli. Our results support that the leucine-specific domain is critical for aminoacylation but not required for editing activity. Kinetic analysis determined that deletion of the LS-domain primarily impacts kcat. Because of its proximity to the aminoacylation active site, we propose that this domain interacts with the tRNA during amino acid activation and/or tRNA aminoacylation. Although the leucine-specific domain does not appear to be important to the editing complex, it remains possible that it aids the dynamic translocation process that moves tRNA from the aminoacylation to the editing complex.     

A comparative study of sulfhydryl groups required for the catalytic activity of gramicidin S synthetase and isoleucyl tRNA synthetase

The sulfhydryl groups required for the catalytic activity of gramicidin S synthetase of Bacillus brevis and Escherichia coli isoleucyl tRNA synthetase were compared. In gramicidin S synthetase 2(GS 2), about four sulfhydryl groups react rapidly with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) or N-ethylmaleimide (NEM), and are essential for gramicidin S formation in the presence of gramicidin S synthetase 1 (GS 1). These sulfhydryl groups are protected against DTNB and NEM reactions by the preincubation of GS 2 with amino acid substrates in the presence of ATP and MgCl2, like the sulfhydryl groups that react rapidly with DTNB or NEM and are required for the catalytic activity of GS 1 and isoleucyl tRNA synthetase. In GS 2, GS 1, and isoleucyl tRNA synthetase, the sulfhydryl group that reacts rapidly with NEM and is required for the catalytic activity is involved in the amino acid binding as a thioester. In isoleucyl tRNA synthetase, it is suggested that isoleucine may be transferred from the isoleucine thioester enzyme complex to tRNA by a mechanism similar to that proposed for gramicidin S synthetase.     

Proofreading in trans by an aminoacyl-tRNA synthetase: a model for single site editing by isoleucyl-tRNA synthetase.

Editing of errors in amino acid selection by an aminoacyl-tRNA synthetase prevents attachment of incorrect amino acids to tRNA, thereby greatly enhancing accuracy of translation of the genetic code. Editing of the non-protein amino acid homocysteine, a frequent type of an error-correcting process, involves reaction of the side chain sulfhydryl group of homocysteine with its activated carboxyl group forming a cyclic thioester, homocysteine thiolactone. Here, it is shown that isoleucyl-tRNA synthetase (IleRS), which occasionally misactivates homocysteine in vitro and in vivo, catalyzes reactions of activated isoleucine with organic thiols (analogues of the side chain of homocysteine). That these enzymatic reactions occur between Ile-tRNAIle or Ile-AMP (bound in the synthetic sub-site) and a thiol (an analogue of the side chain of homocysteine, bound in the editing sub-site), indicates that the two sub-sites are physically close on the surface of IleRS, forming a single synthetic/editing active site of the enzyme. Although IleRS.Val-AMP undergoes thiolysis as efficiently as do IleRS.Ile-AMP and IleRS.Ile-tRNAIle, IleRS.Val-tRNAIle does not react with thiols. These and other data suggest that the mischarged valine residue in IleRS.Val-tRNAIle is, most likely, positioned off the enzyme.     

Cysteinyl-tRNA synthetase is a direct descendant of the first aminoacyl-tRNA synthetase.

The gene encoding the cysteinyl-tRNA synthetase of E. coli was cloned from an E. coli genomic library made in lambda 2761, a lambda vector which can integrate and which carries a chloramphenicol resistance gene. A thermosensitive cysS mutant of E. coli was lysogenised and chloramphenicol-resistant colonies able to grow at 42 degrees C were selected to isolate phages containing the wild-type cysS gene. The sequence of the gene was determined. It codes for a 461 amino-acid protein and includes the sequences HIGH and KMSK known to be involved in the ATP and tRNA binding respectively of class I synthetases. The cysteinyl enzyme has segments in common with the cytoplasmic leucyl-tRNA synthetase of Neurospora crassa, the tryptophanyl-tRNA synthetase of Bacillus stearothermophilus, and the phenylalanyl-tRNA synthetase of Saccharomyces cerevisiae. Sequence comparisons show that the amino end of the cysteinyl-tRNA synthetase has similarities with prokaryotic elongation factors Tu; this region is close to the equivalent acceptor binding domain of the glutaminyl-tRNA synthetase of E. coli. There is a further similarity with the seryl enzyme (a class II enzyme) which has led us to propose that both classes had a common origin and that this was the ancestor of the cysteinyl-tRNA synthetase.     

Aminolevulinate synthase: lysine 313 is not essential for binding the pyridoxal phosphate cofactor but is essential for catalysis.

5-Aminolevulinate synthase is the first enzyme of the heme biosynthetic pathway in animals and some bacteria. Lysine-313 of the mouse erythroid aminolevulinate synthase was recently identified to be linked covalently to the pyridoxal 5'-phosphate cofactor (Ferreira GC, Neame PJ, Dailey HA, 1993, Protein Sci 2:1959-1965). Here we report on the effect of replacement of aminolevulinate synthase lysine-313 by alanine, histidine, and glycine, using site-directed mutagenesis. Mutant enzymes were purified to homogeneity, and the purification yields were similar to those of the wild-type enzyme. Although their absorption spectra indicate that the mutant enzymes bind pyridoxal 5'-phosphate, they bind noncovalently. However, addition of glycine to the mutant enzymes led to the formation of external aldimines. The formation of an external aldimine between the pyridoxal 5'-phosphate cofactor and the glycine substrate is the first step in the mechanism of the aminolevulinate synthase-catalyzed reaction. In contrast, lysine-313 is an essential catalytic residue, because the K313-directed mutant enzymes have no measurable activity. In summary, site-directed mutagenesis of the aminolevulinate synthase active-site lysine-313, to alanine (K313A), histidine (K313H), or glycine (K313G) yields enzymes that bind the pyridoxal 5'-phosphate cofactor and the glycine substrate to produce external aldimines, but which are inactive. This suggests that lysine-313 has a functional role in catalysis.     

Genetic and chemical analyses reveal that trypanothione synthetase but not glutathionylspermidine synthetase is essential for Leishmania infantum

Trypanothione is a unique and essential redox metabolite of trypanosomatid parasites, the biosynthetic pathway of which is regarded as a promising target for antiparasitic drugs. Synthesis of trypanothione occurs by the consecutive conjugation of two glutathione molecules to spermidine. Both reaction steps are catalyzed by trypanothione synthetase (TRYS), a molecule known to be essential in Trypanosoma brucei. However, other trypanosomatids (including some Leishmania species and Trypanosoma cruzi) potentially express one additional enzyme, glutathionylspermidine synthetase (GSPS), capable of driving the first step of trypanothione synthesis yielding glutathionylspermidine. Because this monothiol can substitute for trypanothione in some reactions, the possibility existed that TRYS was redundant in parasites harboring GSPS. To clarify this issue, the functional relevance of both GSPS and TRYS was investigated in Leishmania infantum (Li). Employing a gene-targeting approach, we generated a gsps^−/− knockout line, which was viable and capable of replicating in both life cycle stages of the parasite, thus demonstrating the superfluous role of LiGSPS. In contrast, elimination of both LiTRYS alleles was not possible unless parasites were previously complemented with an episomal copy of the gene. Retention of extrachromosomal LiTRYS in the trys^−/−/+TRYS line after several passages in culture further supported the essentiality of this gene for survival of L. infantum (including its clinically relevant stage), hence ruling out the hypothesis of functional complementation by LiGSPS. Chemical targeting of LiTRYS with a drug-like compound was shown to also lead to parasite death. Overall, this study disqualifies GSPS as a target for drug development campaigns and, by genetic and chemical evidence, validates TRYS as a chemotherapeutic target in a parasite endowed with GSPS and, thus, probably along the entire trypanosomatid lineage.     

Active site of an aminoacyl-tRNA synthetase dissected by energy-transfer-dependent fluorescence.

Aminoacyl-tRNA synthetases establish the rules of the genetic code by catalyzing attachment of amino acids to specific transfer RNAs (tRNAs) that bear the anticodon triplets of the code. Each of the 20 amino acids has its own distinct aminoacyl-tRNA synthetase. Here we use energy-transfer-dependent fluorescence from the nucleotide probe N-methylanthraniloyl dATP (mdATP) to investigate the active site of a specific aminoacyl-tRNA synthetase. Interaction of the enzyme with the cognate amino acid and formation of the aminoacyl adenylate intermediate were detected. In addition to providing a convenient tool to characterize enzymatic parameters, the probe allowed investigation of the role of conserved residues within the active site. Specifically, a residue that is critical for binding could be distinguished from one that is important for the transition state of adenylate formation. Amino acid binding and adenylate synthesis by two other aminoacyl-tRNA synthetases was also investigated with mdATP. Thus, a key step in the synthesis of aminoacyl-tRNA can in general be dissected with this probe.     

tRNA-balanced expression of a eukaryal aminoacyl-tRNA synthetase by an mRNA-mediated pathway

Aminoacylation of transfer RNAs is a key step during translation. It is catalysed by the aminoacyl-tRNA synthetases (aaRSs) and requires the specific recognition of their cognate substrates, one or several tRNAs, ATP and the amino acid. Whereas the control of certain aaRS genes is well known in prokaryotes, little is known about the regulation of eukaryotic aaRS genes. Here, it is shown that expression of AspRS is regulated in yeast by a feedback mechanism that necessitates the binding of AspRS to its messenger RNA. This regulation leads to a synchronized expression of AspRS and tRNA(Asp). The correlation between AspRS expression and mRNA(AspRS) and tRNA(Asp) concentrations, as well as the presence of AspRS in the nucleus, suggests an original regulation mechanism. It is proposed that the surplus of AspRS, not sequestered by tRNA(Asp), is imported into the nucleus where it binds to mRNA(AspRS) and thus inhibits its accumulation.     

Transfer RNA-dependent cognate amino acid recognition by an aminoacyl-tRNA synthetase.

An investigation of the role of tRNA in the catalysis of aminoacylation of Escherichia coli glutaminyl-tRNA synthetase (GlnRS) has revealed that the accuracy of specific interactions between GlnRS and tRNAGln determines amino acid affinity. Mutations in GlnRS at D235, which makes contacts with nucleotides in the acceptor stem of tRNAGln, and at R260 in the enzyme's active site were found to be independent during tRNA binding but interactive for aminoacylation. Characterization of mutants of GlnRS at position 235, showed amino acid recognition to be tRNA mediated. Aminoacylation of tRNA(CUA)Tyr [tyrT (UAG)] by GlnRS-D235H resulted in a 4-fold increase in the Km for the Gln, which was reduced to a 2-fold increase when A73 was replaced with G73. These and previous results suggest that specific interactions between GlnRS and tRNAGln ensure the accurate positioning of the 3' terminus. Disruption of these interactions can change the Km for Gln over a 30-fold range, indicating that the accuracy of aminoacylation is regulated by tRNA at the level of both substrate recognition and catalysis. The observed role of RNA as a cofactor in optimizing amino acid activation suggests that the tRNAGln-GlnRS complex may be partly analogous to ribonucleoprotein enzymes where protein-RNA interactions facilitate catalysis.     

FTIP1 is an essential regulator required for florigen transport

The capacity to respond to day length, photoperiodism, is crucial for flowering plants to adapt to seasonal change. The photoperiodic control of flowering in plants is mediated by a long-distance mobile floral stimulus called florigen that moves from leaves to the shoot apex. Although the proteins encoded by FLOWERING LOCUS T (FT) in Arabidopsis and its orthologs in other plants are identified as the long-sought florigen, whether their transport is a simple diffusion process or under regulation remains elusive. Here we show that an endoplasmic reticulum (ER) membrane protein, FT-INTERACTING PROTEIN 1 (FTIP1), is an essential regulator required for FT protein transport in Arabidopsis. Loss of function of FTIP1 exhibits late flowering under long days, which is partly due to the compromised FT movement to the shoot apex. FTIP1 and FT share similar mRNA expression patterns and subcellular localization, and they interact specifically in phloem companion cells. FTIP1 is required for FT export from companion cells to sieve elements, thus affecting FT transport through the phloem to the SAM. Our results provide a mechanistic understanding of florigen transport, demonstrating that FT moves in a regulated manner and that FTIP1 mediates FT transport to induce flowering.     

A substrate-assisted concerted mechanism for aminoacylation by a class II aminoacyl-tRNA synthetase

Aminoacyl-tRNA synthetases (aaRS) join amino acids to their cognate transfer RNAs, establishing an essential coding relationship in translation. To investigate the mechanism of aminoacyl transfer in class II Escherichia coli histidyl-tRNA synthetase (HisRS), we devised a rapid quench assay. Under single turnover conditions with limiting tRNA, aminoacyl transfer proceeds at 18.8 s(-)(1), whereas in the steady state, the overall rate of aminoacylation is limited by amino acid activation to a rate of 3 s(-)(1). In vivo, this mechanism may serve to allow the size of amino acid pools and energy charge to control the rate of aminoacylation and thus protein synthesis. Aminoacyl transfer experiments using HisRS active site mutants and phosphorothioate-substituted adenylate showed that substitution of the nonbridging Sp oxygen of the adenylate decreased the transfer rate at least 10 000-fold, providing direct experimental evidence for the role of this group as a general base for the reaction. Other kinetic experiments revealed that the rate of aminoacyl transfer is independent of the interaction between the carboxyamide group of Gln127 and the alpha-carboxylate carbon, arguing against the formation of a tetrahedral intermediate during the aminoacyl transfer. These experiments support a substrate-assisted concerted mechanism for HisRS, a feature that may generalize to other aaRS, as well as the peptidyl transferase center.