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© 1996 Oxford University Press 2505-2510

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

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

Department of Microbiology and Molecular Genetics, UMDNJ-New Jersey Medical School, Newark , NJ 07103, USA

Received April 8, 1996; Accepted May 14, 1996

ABSTRACT

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-tRNA Ile 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-tRNA Ile , IleRS - Val-tRNA Ile does not react with thiols. These and other data suggest that the mischarged valine residue in IleRS-Val-tRNA Ile is, most likely, positioned off the enzyme.

INTRODUCTION

Proofreading or editing mechanisms are an essential part of biological information transfer processes, including translation (reviewed in 1 - 3 ). The non-protein amino acid homocysteine (Hcy), an obligatory precursor of methionine in all cells, poses a major accuracy problem for the protein biosynthetic apparatus. Three closely related class I aminoacyl-tRNA synthetases (AARS) (reviewed in 4 ), MetRS, IleRS and LeuRS, misactivate Hcy in vitro ( 5 , 6 ) at a frequency exceeding the frequency of translational errors in vivo ( 2 ). Two other synthetases, class I ValRS ( 5 , 7 ) and class II LysRS (H. Jakubowski, unpublished), misactivate Hcy less efficiently. These five enzymes possess an efficient editing mechanism which prevents misincorporation of Hcy into tRNA ( 5 , 8 ) by destroying the Hcy-AMP intermediate. The editing pathway involves reaction of the side chain sulfhydryl group of Hcy with its activated carboxyl group yielding a cyclic thioester, Hcy thiolactone ( 5 - 7 ). For at least one synthetase, the editing reaction occurs in the same active site that carries out the synthetic reaction, as demonstrated by structure-function studies of MetRS ( 9 ). Editing reactions catalyzed by three class I AARS, MetRS ( 10 - 13 ), IleRS ( 14 ) and LeuRS ( 14 ), have been shown to occur in vivo .

Recently, a class I synthetase, ArgRS, has been shown to catalyze synthesis of dipeptides Arg-Cys and Arg-Hcy from Arg-tRNA Arg and a corresponding thioamino acid ( 15 ). The mechanism involves a nucleophilic attack of the thiolate of cysteine (or homocysteine) on the ester bond in Arg-tRNA Arg . The resulting thioesters S-arginyl-cysteine and S-arginyl-homocysteine are not observed as discrete intermediates because of their rapid rearrangement to form a stable peptide bond. The involvement of thioester intermediates in the synthesis of Arg-Cys and Arg-Hcy is supported by the observation that thioesters of arginine do form with cysteine derivatives that do not have a free amino group, such as N -acetyl-L-cysteine and 3-mercaptopropionate. Although ArgRS does not appear to have an editing mechanism ( 15 ), its ability to catalyze the formation of thioesters of arginine is reminiscent of the formation of the thioester homocysteine thiolactone in proofreading reactions of other AARSs ( 5 - 7 ).

IleRS, which has an editing mechanism directed against Hcy in vivo ( 14 ) is also capable of forming Ile-Cys from Ile-tRNA Ile and cysteine ( 15 ). However, the mechanism of Ile-Cys formation has not been determined and it is not known whether IleRS can also catalyze the formation of thioesters of isoleucine. If it occured, the synthesis of thioesters of isoleucine by IleRS would mimic editing reaction, in which misactivated Hcy is cyclized to the thioester homocysteine thiolactone by the enzyme (see Fig. 4 ). In addition to editing Hcy ( 5 , 14 ), IleRS is also capable of editing valine in vitro ( 16 , 17 ). Editing of valine was suggested to involve a site separate from the synthetic site ( 18 ). It is not known whether noncognate valine and Hcy are edited in the same or separate sites of IleRS. One way to answer this question is to compare reactivities toward thiols of aminacyl ester bonds in cognate IleRS-Ile-tRNA Ile and noncognate IleRS-Val-tRNA Ile complexes. Similar reactivities with thiols of both complexes would suggest that the Val and Ile residues (bound to tRNA Ile ) occupy the same site on the enzyme. If the Val residue in mischarged Val-tRNA Ile occupies a different site, the mischarged tRNA should not react with thiols.

Here, it is shown that IleRS catalyses reactions of cognate Ile-tRNA Ile and Ile-AMP, as well as noncognate Val-AMP, but not Val-tRNA Ile , with organic thiols (analogues of the side chain of Hcy) in reactions mimicking editing of Hcy by the enzyme. The data indicate that two major physiologic functions of IleRS, formation of Ile-tRNA and editing of inadvertently misactivated Hcy, reside in one active site of the enzyme. A misacylated valine residue in an IleRS-Val-tRNA Ile complex, which may form transiently during editing ( 19 ), appears not to be bound on the enzyme but is still hydrolyzed off the tRNA.

MATERIALS AND METHODS

Plasmids and host strain

Plasmids containing the genes for Escherichia coli IleRS ( 18 , 20 ) and Bacillus stearothermophilus ValRS ( 21 ) were obtained from P. Schimmel and E. Schmidt. Plasmids were over-expressed in E.coli strain JM101 and used as a source of AARS. Cells for enzyme purifications were obtained from overnight cultures (usually 400 ml, yielding ~2 g cells) grown at 37oC in LB medium containing 100 [mu]g/ml ampicillin.

Aminoacyl-tRNA synthetases

Escherichia coli IleRS ( 20 ) and B.stearothermophilus ValRS ( 21 , 22 ) were purified to homogeneity from the overproducing strains using standard procedures ( 20 , 22 ).

Preparation of [ 14 C]Ile-tRNA Ile and [ 14 C]Val-tRNA Ile

[ 14 C]Ile-tRNA Ile was prepared from aminoacylation mixtures (0.1 ml) containing 50 mM HEPES, pH 7.4, 10 mM MgCl 2 , 0.1 mM EDTA, 2.5 mM ATP, 10 [mu]M tRNA Ile from E.coli (1600 pmol/ A 260 , Subriden RNA), 45 [mu]M [ 14 C]isoleucine (306 Ci/mol) (NEN) and 0.1 [mu]M E.coli IleRS. [ 14 C]Val-tRNA Ile was prepared by mischarging ( 22 ) E.coli tRNA Ile with 25 [mu]M [ 14 C]valine (285 Ci/mol) (Amersham) using B.stearothermophilus ValRS ( 18 , 21 , 22 ). After 15 min at 37oC the charged tRNA was purified by phenol extraction, and recovered by precipitation with ethanol. The precipitate was washed several times with 70% ethanol to remove traces of free [ 14 C]amino acids, dissolved in 0.1 ml glass-distilled water, and stored at -20oC.

Enzymatic deacylation of [ 14 C]aminoacyl-tRNA Ile

The reactions were carried out at 37oC in 0.1 M K-HEPES, pH 7.4, 10 mM MgCl 2 , 0.2 mM EDTA. In one set of experiments, the disappearance of [ 14 C]aminoacyl-tRNA Ile was monitored by trichloroacetic acid precipitation. In another set of experiments in which all forms of [ 14 C]amino acids were followed, the aliquots were analyzed by TLC.

Thiolysis of IleRS - [ 14 C]aminoacyl-AMP

IleRS-[ 14 C]Ile-AMP was prepared and incubated with thiols in the same mixture. The reactions were carried out at 37oC in mixtures containing 1 [mu]M Ile-RS, 15 [mu]M [ 14 C]isoleucine, 1 mM ATP, 0.1 M K-HEPES, pH 7.4, 10 mM MgCl 2 , 0.2 mM EDTA, 50 mM thiol and 5 U/ml inorganic pyrophosphatase (Sigma). The reactions were monitored by TLC. Under these conditions, reactions with L-cysteine, cysteamine and dithiothreitol (DTT) were completed within 2 min, and with D,L-Hcy in 16 min. With 2-mercaptoethanol (2-ME), reactions were 50% completed in 16 min. Similar reactions were carried out with IleRS-[ 14 C]Val-AMP prepared in reaction mixtures containing 15 or 150 [mu]M [ 14 C]valine instead of [ 14 C]isoleucine.


Figure 1 . Effects of thiols and hydroxylamine on the rate of enzymatic deacylation of Ile-tRNA Ile . Reactions were carried out at 37oC in reaction mixtures containing 0.1 M HEPES-KOH (pH 7.4), 10 mM MgCl 2 , 0.1 mM EDTA, 0.8 [mu]M [ 14 C]Ile-tRNA Ile (1 pmol = 400 c.p.m.), 1 [mu]M IleRS and L-cysteine, DTT or hydroxylamine. First order rate constants, k , are plotted as a function of concentrations of indicated compounds.

TLC analysis

TLC separations were carried out on cellulose plates from Kodak using butanol-acetic acid-water (4:1:1, v/v) as a solvent ( 5 , 15 , 23 ). Authentic isoleucine and valine (Sigma) standards were co-chromatographed with samples and visualized by staining with ninhydrin. TLC plates were autoradiographed using Reflectiontm (NEN) autoradiography film.

RESULTS AND DISCUSSION

Some thiols accelerate enzymatic deacylation of Ile-tRNA Ile

Most AARS ( 2 , 3 , 23 ), including IleRS ( 19 , 24 , 25 ), catalyze deacylation reactions which result in discharging of an amino acid from aminoacyl-tRNA in the absence of AMP and PP i . As shown in Table 1 , enzymatic deacylation of Ile-tRNA was accelerated by L-cysteine and DTT but not by D-cysteine, D,L-Hcy and 2-ME, indicating specificity of the reaction with respect to which thiols can participate. This also suggests that stimulation is not merely due to reactivation of essential -SH groups of the enzyme. When reactions were carried out at different thiol concentrations, saturation kinetics were observed (Fig. 1 ). Control experiments have shown that L-cysteine and DTT did not stimulate nonenzymatic deacylation of Ile-tRNA. The half-life for Ile-tRNA Ile (pH 7.4, 37oC) in the absence and presence of 0.125 M L-cysteine or DTT was 180 min. Another nucleophile, hydroxylamine, did not accelerate the enzymatic deacylation (Fig. 1 ), but did stimulate nonenzymatic deacylation of Ile-tRNA Ile (the half-life for Ile-tRNA Ile was 110 min in the presence of 125 mM hydroxylamine). These results suggest the presence on the enzyme of a specific site (`-SH subsite'), which affects enzymatic deacylation of Ile-tRNA.

Thiols react enzymatically with Ile-tRNA Ile

To analyze products of enzymatic deacylation of Ile-tRNA, aliquots of reaction mixtures containing IleRS, [ 14 C]Ile-tRNA Ile and various thiols were subjected to TLC. In the absence of thiols, isoleucine was a major product (lane 1, Fig. 2 A, B and C). In the presence of L-cysteine (lane 4, Fig. 2 A; lane 2, Fig. 2 B), DTT (lane 3, Fig. 2 A), cysteamine (lane 5, Fig. 2 A; lane 3, Fig. 2 B), 3-mercaptopropionate, N- acetyl-L-cysteine, L-cysteine methyl ester (lanes 4, 7 and 8 respectively, Fig. 2 B), and D-cysteine (lane 7, Fig. 2 A), new products formed, suggesting that these thiols bind to the -SH subsite and react with Ile-tRNA. Deacylations in the presence of L-cysteine (lane 4, Fig. 2 A; lane 2, Fig. 2 B) and D,L-Hcy (lane 6, Fig. 2 A) also yielded isoleucine. These observations indicate that, in addition to binding to the -SH subsite in the editing mode common for all thiols (see Fig. 4 B.iv), L-cysteine and Hcy bind also in the synthetic mode as substrates ( 5 ) with their side chain -SH unable to react with Ile-tRNA Ile (Fig. 4 A.i). Cysteine, in addition to Hcy, is a noncognate substrate which is misactivated and efficiently edited by IleRS ( 5 ). Control reactions showed that alanine (lane 5, Fig. 2 B), serine (lane 9, Fig. 2 B) and S -methyl-L-cysteine (lane 6, Fig. 2 B) did not react with Ile-tRNA Ile .


Figure 2 . TLC analysis of products of enzymatic deacylation of Ile-tRNA Ile . Reactions mixtures were incubated at 37oC for 5 min and spotted on the origin line of cellulose (A and C) or silica gel (B) TLC plates (Merck). The plates were developed at a distance of 10 cm with butanol-acetic acid-water (4:1:1, v/v) as solvent. Autoradiograms exposed from these plates are shown. The following compounds were tested at 20 mM. ( A) Lane 1, no additions; lane 2, 2-ME; lane 3, DTT; lane 4, L-cysteine; lane 5, cysteamine; lane 6, D,L-Hcy; lane 7, D-cysteine. ( B) Lane 1, no additions; lane 2, L-cysteine; lane 3, cysteamine; lane 4, 3-mercaptopropionate; lane 5, alanine; lane 6, S -methyl-L-cysteine; lane 7, N -acetyl-L-cysteine; lane 8, L-cysteine methyl ester; lane 9, serine. ( C) Competition between isoleucine and thiols. Reaction mixtures containing 100 mM L-cysteine or DTT, and/or 2.5 mM isoleucine were analyzed: lane 1, no additions; lane 2, isoleucine; lane 3, L-cysteine; lane 4, L-cysteine + isoleucine; lane 5, DTT; lane 6, DTT + isoleucine. Spots migrating close to the origin (occupied by Ile-tRNA spot) in lanes 4, 5 and 7 in (A), lanes 2, 3 and 8 in (B) are oxidation products (disulfides) of the fastest migrating spots.

Identification of products of thiol-dependent deacylation of Ile-tRNA Ile

To determine identities of these new products, several chemical tests were carried out followed by TLC analysis of treated and untreated samples. For example, products of enzymatic reactions of Ile-tRNA with N- acetyl-L-cysteine, 3-mercaptopropionate and DTT were hydrolyzed to isoleucine upon NaOH treatment and, with the exception of the DTT-dependent product, were not sensitive to thiol reagents such as iodoacetate and DTNB. These properties suggest that products formed in the presence of N- acetyl-cysteine, 3-mercaptopropionate and DTT were the corresponding thioesters of isoleucine.

In contrast, products of enzymatic reaction of Ile-tRNA with cysteamine, D-cysteine, L-cysteine and L-cysteine methyl ester were not sensitive to NaOH but reacted with iodoacetate and DTNB. Upon Raney nickel treatment (which desulphurizes cysteine into alanine), a product of the IleRS-dependent reaction of Ile-tRNA with cysteine was transformed into a new product which co-chromatographed with an authentic Ile-Ala standard ( 15 ). These properties suggest that products of cysteamine-, D-cysteine-, L-cysteine- and L-cysteine methyl ester-dependent enzymatic deacylation of Ile-tRNA Ile are the corresponding isoleucyl-dipeptides.

Table 1 . First order rate constants, k , for enzymatic deacylation of Ile-tRNA Ile and Val-tRNA Ile catalyzed by IleRS Val-tRNA Ile
Additions

k (s -1 )

Ile-tRNA Ile

None

0.04

0.8

Valine

N.D.

0.6

D,L-Hcy

0.04

0.6

L-Cysteine

0.08

0.5

Isoleucine (2.5 mM)

0.04

0.8

Cysteine + isoleucine (2.5 mM)

0.04

N.D.

DTT

0.08

0.5

DTT + isoleucine (2.5 mM)

0.04

N.D.

2-ME

0.04

N.D.

D-Cysteine

0.04

N.D.

Deacylations were carried out at 37oC in reaction mixtures containing 100 mM HEPES-KOH (pH 7.4), 10 mM MgCl 2 , 0.1 mM EDTA, 1.4 [mu]M [ 14 C]Ile-tRNA Ile (1 pmol = 400 c.p.m.) or 1.1 [mu]M [ 14 C]Val-tRNA Ile (1 pmol = 350 c.p.m.), 0.2 or 0.025 [mu]M IleRS and indicated additions at 50 mM unless indicated otherwise. The k values (calculated as in ref. 15) are means of two to three independent determinations with errors of 20%. N.D., not determined.

Formation of isoleucyl-dipeptides is consistent with the following mechanism. The ester bond in Ile-tRNA Ile undergoes thiolation by cysteine (or similar thiol amino acid). The resulting thioester is not observed as an intermediate because of the rapid rearrangement to form a stable peptide bond. Facile intramolecular reaction results from the favorable geometric arrangement of the [alpha]-amino group of cysteine with respect to the thioester bond ( 26 ). Support for this mechanism comes from the observations that the -SH group is required for the reaction (alanine, serine and S- methyl-cysteine do not react with Ile-tRNA Ile , Fig. 2 B) and that isoleucine thioesters do indeed form with cysteine derivatives that do not have a free amino group, such as 3-mercaptopropionate and N -acetyl-L-cysteine. A similar mechanism accounts for synthesis of the dipeptide Arg-Cys by Arg-RS ( 15 ).

Products of nonenzymatic deacylation of Ile-tRNA in the presence of cysteine and DTT were also analyzed by TLC. The half life of Ile-tRNA Ile (180 min) was not affected by thiols as stated above. More than 95% isoleucine and <5% Ile-Cys or Ile-DTT formed during these nonenzymatic reactions. These results further indicate that reactions of thiols with Ile-tRNA Ile are catalyzed by IleRS.

Thiols bind at a distinct site of IleRS

To test whether binding sites for isoleucine and thiols interact, thiol-dependent deacylation reactions were carried out in the absence and presence of exogenous isoleucine. The cysteine- and DTT-dependent deacylations of Ile-tRNA Ile were 2-fold slower in the presence of isoleucine than in its absence (Table 1 ). As shown in Figure 2 C, exogenous isoleucine prevented formation of [ 14 C]Ile-DTT (thio)ester (compare lanes 6 and 5) and [ 14 C]Ile-Cys dipeptide (compare lanes 4 and 3) from [ 14 C]Ile-tRNA Ile . This suggests that free isoleucine displaces the [ 14 C]Ile residue in [ 14 C]Ile-tRNA Ile from the thiol-reactive site on IleRS either to another site on the enzyme (perhaps the editing site for valine) or off the enzyme. Because [ 14 C]Ile-tRNA Ile is deacylated by IleRS even in the presence of free isoleucine or valine ( 24 ; see also Table 1 ), it follows that under these conditions the [ 14 C]Ile residue in an [ 14 C]Ile-tRNA Ile -IleRS complex is positioned off the enzyme: excess of free isoleucine or valine is expected to prevent binding of the [ 14 C]Ile residue in [ 14 C]Ile-tRNA Ile -IleRS to the enzyme. Alternatively, free isoleucine might bind to a second Ile-binding site which in turn would either displace a thiol or affect reactivity of the ester bond in IleRS-bound Ile-tRNA Ile . However, IleRS, a monomeric enzyme ( 20 ), is very unlikely to possess two Ile-binding sites. That thiols do not bind at the isoleucine binding site of IleRS is indicated by the inability of thiols to inhibit the aminoacylation reaction catalyzed by IleRS (see below).

Thiols and valine do not affect deacylation of Val-tRNA Ile by IleRS

The rate of IleRS-dependent deacylation of [ 14 C]Val-tRNA Ile , in contrast with the rate of deacylation of [ 14 C]Ile-tRNA Ile , was not accelerated by DTT or L-cysteine (Table 1 ). Saturating concentrations of Hcy and valine, noncognate substrates which are misactivated and edited by IleRS ( 5 , 16 ), did not prevent enzymatic deacylation of [ 14 C]Val-tRNA Ile (Table 1 ). TLC analyses demonstrated that [ 14 C]valine was the only product of the deacylation reaction in the presence or absence of 100 mM L-cysteine, D,L-Hcy and DTT (not shown). The sensitivity of these analyses was such that <= 5% of Val-Cys or Val-DTT would have been detected if present. These results suggest that the charged valine and isoleucine residues in Val-tRNA Ile -IleRS and Ile-tRNA Ile -IleRS complexes respectively, are positioned differently. The [ 14 C]isoleucine residue in [ 14 C]Ile-tRNA Ile -IleRS is positioned on the enzyme and therefore able to react with thiols; excess of free isoleucine or valine displaces the [ 14 C]isoleucine to the off enzyme position thereby preventing reaction with thiols.

The valine residue in Val-tRNA Ile -IleRS is, most likely, positioned off the enzyme and is therefore unreactive towards thiols. Because neither exogenous valine nor isoleucine significantly inhibits deacylation of Val-tRNA Ile by IleRS (Table 1 ), it is unlikely that the valine residue in Val-tRNA Ile -IleRS is positioned on the enzyme. A proposed second site for editing of mischarged valine by IleRS ( 18 ) would be functionally equivalent to the off enzyme position.

Reactions of IleRS  Ile-AMP and IleRS  Val-AMP with thiols

Thiols reacted also with IleRS-bound Ile-AMP or Val-AMP, yielding thioesters and peptides. For example, IleRS-Ile-AMP reacted with DTT and 2-ME to yield (thio)esters Ile-DTT and Ile-(2-ME). Similarly, IleRS-Val-AMP reacted with DTT to yield (thio)ester Val-DTT. Peptide bond formation occurred with cysteine to yield either Ile-Cys or Val-Cys. The reactions with adenylates proceeded at about the same rates as enzymatic reactions with Ile-tRNA Ile and exhibited similar thiol specificity: cysteine and DTT reacted faster than 2-ME (not shown). Similar reactivities of IleRS-bound aminoacyl-adenylates and Ile-tRNA Ile towards thiols indicate that the amino acid residues of Ile-AMP, Val-AMP and Ile-tRNA Ile occupy the same sub-site in the active site of the enzyme.


Figure 3 . Effects of thiols on aminoacylation of tRNA Ile . Reactions were carried out at 37oC in mixtures containing 0.1 M HEPES-KOH (pH 7.4) buffer, 15 [mu]M tRNA Ile , 16 [mu]M [ 14 C]Ile (306 Ci/mol) 2.5 mM ATP, 10 mM KF, 10 mM MgCl 2 and 30 nM IleRS. Time courses of aminoacylation in the absence and presence of 20 mM ( A ) and 200 mM ( B ) indicated thiol are shown. ( C) TLC analysis of aminoacylation mixtures containing 200 mM (lanes 1-3) or 20 mM (lanes 5-9) thiol: lanes 1 and 5, DTT, lanes 2 and 6, 2-ME; lanes 3 and 9, D-cysteine; lane 4, no additions; lane 7, D,L-Hcy; lane 8, L-cysteine. Spots migrating close to Ile-tRNA Ile in lanes 3, 8 and 9 are disulfide forms of Ile-Cys dipeptide.


The (thio)esters [ 14 C]Ile-DTT and [ 14 C]Val-DTT can be easily purified by extraction of reaction mixtures with toluene. TLC analysis showed that mild alkaline hydrolysis of [ 14 C]Ile-DTT yields [ 14 C]isoleucine, whose identity was confirmed by showing that it can re-charge tRNA Ile in the presence of ATP and IleRS. Similarly, mild alkaline hydrolysis of [ 14 C]Val-DTT yielded [ 14 C]valine, whose identity was confirmed by showing that it can re-charge tRNA Val in the presence of ATP and ValRS. The half-life of the spontaneous hydrolysis of both [ 14 C]Ile-DTT and [ 14 C]Val-DTT at pH 7.4, 37oC was 120 min, somewhat less than the half-life of Ile-tRNA Ile (180 min), Val-tRNA Ile (160 min) or Val-tRNA Val (140 min).


Figure 4 . A model for single site editing by IleRS . The active site is proposed to have two partially overlapping subsites, synthetic and editing. The [alpha]-carbon, carboxyl and amino groups, common to [alpha]-amino acids, bind to the overlapping region common to the synthetic and editing subsites. The side chain of an amino acid can bind to nonoverlapping portions of either synthetic or editing subsite. Thus, an amino acid binds either in the synthetic or editing mode. Amino acid substrates of IleRS bind initially in the synthetic mode. Binding of a noncognate amino acid in the editing mode is induced at some point of the synthetic pathway. ( A) (i) Initial binding of the noncognate Hcy is in the synthetic subsite of IleRS. (ii) The side chain of Hcy moves to the editing subsite after formation of Hcy-AMP. Nucleophilic attack of the side chain thiol on activated carboxyl carbon yields Hcy thiolactone (iii). ( B) With the synthetic subsite occupied by the cognate isoleucine, the editing subsite can be filled with an analogue of the side chain of Hcy, R-CH 2 -SH (an organic thiol). (iv) This leads to formation of a thioester of isoleucine (v) in a reaction mimicking editing of Hcy. X denotes tRNA Ile or AMP.

Thiols react with isoleucine in complete aminoacylation mixtures

Reactions of thiols with isoleucine occurred also in complete aminoacylation mixtures (Fig. 3 C). This led to transient formation of Ile-tRNA followed by its enzymatic thiolysis in reaction mixtures containing 20 mM L-cysteine or DTT (Fig. 3 A) or 200 mM D-cysteine (Fig. 3 B). Although 2-ME reacted with isoleucine in complete aminoacylation mixtures to some extent (compare lanes 2 and 6 in Fig. 3 C), the reaction was very inefficient and did not lead to transient aminoacylation kinetics (Fig. 3 A and B). Importantly, the rate of the aminoacylation reaction was not inhibited by thiols, even when present at 0.2 M (Fig. 3 A and B), indicating that thiols and isoleucine bind to different (sub)sites on the enzyme.

A model for single site editing of Hcy by IleRS

The ability of IleRS to catalyze synthesis of thioesters of isoleucine is a consequence of the editing function of the enzyme and can be rationalized by the following model (Fig. 4 ). When misactivated Hcy is in the active site of IleRS, its side chain -SH group occupies a sub-site (an -SH sub-site) next to its carboxyl carbon (Fig. 4 A.ii). This sub-site is presumed to be vacant when a cognate amino acid is in the active site. Filling the -SH sub-site by providing the -SH function in trans , i.e., on another molecule (Fig. 4 B.iv), leads to the formation of a thioester of a cognate amino acid (Fig. 4 B.v.). That this reaction occurs between Ile-tRNA Ile or Ile-AMP (bound in the synthetic sub-site) and an organic thiol, an analogue of the side chain of Hcy (bound in the editing sub-site), indicates that the two sub-sites are intimately close on the surface of the enzyme, forming a single synthetic/editing active site. Editing of valine can also be accommodated in the same site: however, while a mischarged valine residue (attached to tRNA Ile ) that undergoes editing appears to be positioned off the surface of the enzyme, the aminoacyl bond is still accesssible to hydrolysis by the enzyme. Thus, editing occurs when an activated amino acid is no longer bound in the synthetic sub-site or when the editing sub-site is occupied by a thiol.

The model shown in Figure 4 is also consistent with the data obtained by others ( 18 ). Implication of this model is that any mutation that affects binding of the side chain of the amino acid substrate during synthetic reaction will not affect editing reaction: the side chain of an amino acid that undergoes editing is not bound in the synthetic sub-site. This explains the behavior of a G56A mutant of IleRS that has lost the ability to discriminate between isoleucine and valine in the synthetic reaction (mostly due to the loss of efficient binding of the side chain of the substrate isoleucine) but was completely active in editing of valine. A related mutant, G56P, inactive in the synthetic reaction with either isoleucine or valine, was active in editing of valine ( 18 ). The lack of synthetic activity of the G56P IleRS mutant is, most likely, due to a total loss of the ability to bind amino acid substrates; this, according to the model shown in Figure 4 , will not affect editing activity of the enzyme. Another mutant IleRS, F570S, has lost the ability to efficiently bind isoleucine and valine during the synthetic reaction, but was still unimpaired in the editing reaction ( 18 ), again consistent with the model shown in Figure 4 .

ACKNOWLEDGEMENTS

I thank Paul Schimmel and Eric Schmidt for plasmids containing the genes for IleRS and ValRS. This work was supported by grants from the American Cancer Society (NP-904) and National Science Foundation (MCB-9218358). I am grateful to Manny Goldman for his critical discussions.

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