氨酰-tRNA合成酶对tRNA的识别

氨酰-tRNA合成酶(aaRS)与tRNA的相互作用保证了蛋白质生物合成的忠实性. 氨酰-tRNA合成酶对tRNA识别的专一性依赖于aaRS特定的催化结构域和tRNA分子特异的三级结构构象. 反密码子和接受茎(包括73位)在大多数aaRS对tRNA分子的识别过程中起着关键作用, 其他部位如可变口袋、可变(茎)环等, 甚至修饰核苷酸对于一些识别过程也有重要作用.     

一种插入116个氨基酸的亮氨酰—tRNA合成酶具有高酶活力

大肠杆菌亮氨酰 tRNA合成酶 (LeuRS)是第 1类氨基酰 tRNA合成酶 ,由 860个氨基酸残基组成 ,催化亮氨酸tRNA的亮氨酰化。研究发现 ,在它的CP1结构域内 3 68和 3 69间的肽键间插入 2 5 3～ 3 68的肽段 ,该插入变种的酶仍具有酶活力 ,取名为LeuRS C。由于这一插入变种的不稳定性 ,构建了His6 LeuRS C的表达质粒 ,用Ni NTA柱亲和层析的方法进行纯化。发现His6 LeuRS C虽然插入了 116个氨基酸残基 ,但仍具有全部的天然LeuRS的活力。测定了His6 LeuRS C的酶学动力学常数 ,比较了它与天然LeuRS的从CD光谱得到的二级结构和热稳定性     

氨酰tRNA合成酶的分子网络和功能

氨酰tRNA合成酶是生命进化过程中最早出现的一类蛋白质,氨酰tRNA合成酶帮助氨基酸转移到相应的tRNA上,进而参与蛋白质的合成保证了生命体的严谨性和多样性.随着后基因组时代的到来,氨酰tRNA合成酶的结构和功能成为新的研究热点.结构生物学和生物信息学的研究结果表明,氨酰tRNA合成酶在真核生物体内以多聚复合物的形式行使功能,形成复杂的分子网络体系.最新的实验证据显示,氨酰tRNA合成酶不但是蛋白质合成过程中一类最重要的酶,而且参与了转录、翻译水平的调控、RNA剪接、信号传导和免疫应答等众多生命活动.     

氨基酰-tRNA合成酶与神经退行性疾病

氨基酰-tRNA合成酶催化tRNA的氨基酰化反应为生物体内的蛋白质合成提供原料.这类古老且保守的蛋白质分子在高等生物复杂的细胞分子网络中分化出的新功能是目前人们关注的焦点.近期在对一些患有神经退行性疾病的病人和小鼠模型的研究中发现,位于酪氨酰-tRNA合成酶、甘氨酰-tRNA合成酶和丙氨酰-tRNA合成酶上的突变,可分别导致DI腓骨肌萎缩症(Charcot-Marie-Toothdisease,CMT)C型,腓骨肌萎缩症2D型及小脑浦肯雅(Purkinje)细胞丢失.初步的致病机理研究表明,致病突变对这3种酶的影响各不相同:酪氨酰-tRNA合成酶的氨基酰化催化能力受到影响,甘氨酰-tRNA合成酶受影响的可能是一种未知的新功能,而丙氨酰-tRNA合成酶受影响的则是它的编校功能.这些研究结果揭示了氨基酰-tRNA合成酶涉及神经退行性疾病的广泛性和其机制的复杂性,并将促进对神经退行性疾病这一类常见疾病的病理和治疗方法的研究.     

以布氏锥虫亮氨酰tRNA合成酶为靶标的抗锥虫药物筛选系统的建立

锥虫是人的血液寄生虫,对热带乃至拉丁美洲贫困地区影响极大,但目前的传统治疗药物存在副作用大、有效性不断降低的问题。根据亮氨酰tRNA合成酶在微生物中可作为药物靶点的事实,在新型抗锥虫药物筛选中,通过对锥虫的亮氨酰tRNA合成酶的克隆、表达和纯化,以及该酶活性测定的优化,建立了该酶抑制物的筛选系统。筛选结果表明,这一以锥虫亮氨酰tRNA合成酶为靶标的抗锥虫药物筛选系统可以有效筛选抗锥虫化合物,选出的化合物有一定的抗锥虫特异性,并可以用于化合物的进一步优化和测定其半抑制浓度。使用这一系统筛选到了对锥虫有较好抑制作用,但对人类细胞毒性较小的一系列新型化合物,因而锥虫亮氨酰tRNA合成酶很可能成为开发有效抗锥虫药物的新靶标。     

蓖麻蚕氨基酰-tRNA合成酶与丝心蛋白合成的关系

蓖麻蚕Phtlosamta cynthia ricim后部丝腺体在五龄后期主要合成一种蛋白质——丝心蛋白。在组成丝心蛋白的氨基酸成分中,丙氨酸和甘氨酸占了78％,其中内氨酸的含量比甘氨酸高(Kirimura等1962)。已经知道在五龄期,蓖麻蚕后部丝腺体tRNA含量与丝心蛋白的氨基酸成分之间存在着相关性(辜祥荣等,1981)。在生物体内,氨基酸必须与其相应的tRNA结合才能参与蛋白质的合成,而两者的结合反应是由其相应的氨基酰-tRNA合成酶所催化的。本文测定了五龄期蓖麻蚕后部丝腺体中甘氨酰-tRNA合成酶和丙氨酰-tRNA合成酶的活力,以及这两个酶的Km值,观察到在五龄期,这两个酶的活力是随着蛋白质合成的增加而升高的,而且并没有同功酶或酶的变构产生。     

精胺对牛肝tRNA~(lle)氨基酰化的刺激作用

本实验用纯化的牛肝异亮氨酸tRNA(tRNA~(Ile))和异亮氨酰tRNA合成酶(IleRS),研究了精胺对Ile-tRNA复合物形成及IleRS活性的作用。结果表明:精胺能特异地促使牛肝tRNA~(Ile)氨基酰化反应;对IleRS活性无影响;能明显地增加形成Ile-tRNA复合物反应的Vmax和tRNA~(Ile)的Km值。     

水稻线粒体tRNATrp突变体的克隆和氨酰化活力鉴定

为了研究tRNATrp的氨基酸接受茎中除两对半碱基以外的特异性元件,设计并完成了4种水稻线粒体tRNATrp向枯草杆菌tRNATrp的突变体(MPB0,G1A和U5G/A68C;MPB1,C2G/G71C:MPB2,C4G/G69C;MPB3,C2G/G71C和C4G/G69C),体外转录并用枯草杆菌和人这两种不同种属来源的色氨酰tRNA合成酶(TrpRS)测定了这些tRNATrp分子的氨酰化活力(Kcat/KM.结果表明,这些突变体具有被枯草杆菌TrpRS氨酰化的能力,与野生型水稻线粒体tRNATrp>相比,MPB0被枯草杆菌TrpRS氨酰化的活力提高了5倍,MPB1和MPB2被枯草杆菌TrpRS氨酰化的活力分别提高了40和53倍,MPB3则提高了140倍,为野生型枯草杆菌tRNATrp>的34%,而人色氨酰tRNA合成酶氨酰化这4个突变体的活力都很微弱.揭示了水稻线粒体tRNATrp>氨基酸接受茎上的2个碱基对C2/G71和C4/G69的突变,对枯草杆菌TrpRS的识别起重要作用,由此推测,接受茎上的2个碱基对C2/G71和C4/G69也是线粒体tRNATrp>重要的特异性元件.     

3种水稻线粒体tRNA~(Trp)突变体的克隆和活力鉴定

 设计并完成了 3种水稻线粒体tRNATrp的突变 ,体外转录并用枯草杆菌和人色氨酰tRNA合成酶 (TrpRS)对tRNATrp及其突变体进行了活力测定 .3种突变体的氨酰化活力比野生型水稻线粒体tRNATrp分别上升了 1 8、1 5和 5倍 .说明A1 U72和G5 C68对于提高线粒体tRNATrp被细胞质TrpRS氨酰化能力的作用并不大 ,细胞质tRNATrp与细胞质TrpRS的识别方式并不适用于线粒体tRNATrp与细胞质TrpRS的相互识别 .研究结果对于了解线粒体tRNATrp和细胞质TrpRS的相互识别及药物设计有重要意义     

氨酰tRNA 合成酶抑制剂作为新型抗感染药物的研究进展

细菌耐药性的不断上升对现有阶段的抗生素类药物提出了一个严峻的挑战,同时也掀起了针对于新靶标的抗菌药物的研究。氨酰tRNA合成酶(aaRS)催化特定氨基酸连接到相应的tRNA分子上,在蛋白质的合成过程中起着必不可少的作用。氨酰tRNA合成酶的抑制会导致蛋白质合成的停止,扰乱细菌和真菌的生长,因此氨酰tRNA合成酶是一类潜在的抗感染靶标。本文分别综述了天然产物及其衍生的aaRS抑制剂,底物和反应中间体模拟物,通过合成和通过虚拟筛选得到的aaRS抑制剂作为新型抗细菌和抗真菌药物的研究进展,并对aaRS的靶标特点、分类和催化机制作一简要介绍。     

Sequence, structural and evolutionary relationships between class 2 aminoacyl-tRNA synthetases

Class 2 aminoacyl-tRNA synthetases, which include the enzymes for alanine, aspartic acid, asparagine, glycine, histidine, lysine, phenylalanine, proline, serine and threonine, are characterised by three distinct sequence motifs 1,2 and 3 (reference 1). The structural and evolutionary relatedness of these ten enzymes are examined using alignments of primary sequences from prokaryotic and eukaryotic sources and the known three dimensional structure of seryl-tRNA synthetase from E. coli. It is shown that motif 1 forms part of the dimer interface of seryl-tRNA synthetase and motifs 2 and 3 part of the putative active site. It is further shown that the seven alpha 2 dimeric synthetases can be subdivided into class 2a (proline, threonine, histidine and serine) and class 2b (aspartic acid, asparagine and lysine), each subclass sharing several important characteristic sequence motifs in addition to those characteristic of class 2 enzymes in general. The alpha 2 beta 2 tetrameric enzymes (for glycine and phenylalanine) show certain special features in common as well as some of the class 2b motifs. In the alanyl-tRNA synthetase only motif 3 and possibly motif 2 can be identified. The sequence alignments suggest that the catalytic domain of other class 2 synthetases should resemble the antiparallel domain found in seryl-tRNA synthetase. Predictions are made about the sequence location of certain important helices and beta-strands in this domain as well as suggestions concerning which residues are important in ATP and amino acid binding. Strong homologies are found in the N-terminal extensions of class 2b synthetases and in the C-terminal extensions of class 2a synthetases suggesting that these putative tRNA binding domains have been added at a later stage in evolution to the catalytic domain.     

Structure and evolution of a group of related aminoacyl-tRNA synthetases

A yeast nuclear gene, designated MSK1, has been selected from a yeast genomic library by transformation of a respiratory deficient mutant impaired in acylation of mitochondrial lysine tRNA. This gene confers a respiratory competent phenotype and restores the mutant's ability to acylate the mitochondrial lysine tRNA. The amino acid sequence of the protein encoded by MSK1 is homologous to yeast cytoplasmic lysyl-tRNA synthetase and to the product of the herC gene, which has recently been suggested to code for the Escherichia coli enzyme. These observations indicate that MSK1 codes for the lysyl-tRNA synthetase of yeast mitochondria. Several regions of high primary sequence conservation have been identified in the bacterial and yeast lysyl-tRNA synthetases. These domains are also present in the aspartyl- and asparaginyl-tRNA synthetases, further confirming the notion that all three present-day enzymes originated from a common ancestral gene. The most conserved domain, located near the carboxyl terminal ends of this group of synthetases is characterized by a cluster of glycines and is also highly homologous to the carboxyl-terminal region of the E. coli ammonia-dependent asparagine synthetase. A catalytic function of the carboxyl terminal domain is indicated by in vitro mutagenesis of the yeast mitochondrial lysyl-tRNA synthetase. Replacement of any one of three glycine residues by alanine and in one case by aspartic acid completely suppresses the activity of the enzymes, as evidenced by the inability of the mutant genes to complement an msk1 mutant, even when present in high copy. Other mutations result in partial loss of activity. Only one glycine replacement affects the stability of the protein in vivo. The observed presence of a homologous domain in asparagine synthetase, which, like the aminoacyl-tRNA synthetases, catalyzes the formation of an aminoacyladenylate, suggests that the glycine-rich sequence is part of a catalytic site involved in binding of ATP and of the aminoacyladenylate intermediate.     

Role of 5SrRNA as a positive effector of some aminoacyl-tRNA synthetases in macromolecular complexes, with specific reference to methionyl-tRNA synthetase.

1) Rat liver 5SrRNA enhanced the activity of methionyl-tRNA synthetase in the macromolecular aminoacyl-tRNA synthetase complex (Fraction B) purified from a rat liver supernatant. 5SrRNA-L5 protein complexes (5SrRNP) had similar effects, whereas other ribosomal RNAs and E. coli 5SrRNA had no effect. 2) 5SrRNA increased the activity of the complex for methionine-dependent ATP-PPi exchange. 3) 5SrRNA increased the activities of methionyl-, arginyl-, and isoleucyl-tRNA synthetases in the complex, but scarcely affected its leucyl-, lysyl-, and glutamyl-tRNA synthetase activities. 4) 5SrRNA increased the activities of the rat liver supernatant for the attachment of [35S]methionine, [3H]isoleucine, [3H]lysine, [3H]proline, [3H]threonine, [3H]tyrosine, and [3H]phenylalanine to endogenous tRNA markedly, and those for [3H]leucine, [3H]arginine, [3H]aspartic acid, and [3H]histidine slightly, but did not affect those for [3H]glutamic acid, [3H]glycine, [3H]valine, [3H]alanine, and [3H]tryptophan. 5) Preincubation of the rat liver supernatant with an antibody against Artemia salina ribosomal protein L5, that cross-reacted with the rat liver ribosomal protein L5, decreased the attachment of [35S]methionine and [3H]isoleucine to endogenous tRNA, and 5SrRNA and 5SRNP enhanced these activities of the supernatant preincubated with antibody. On the other hand, the antibody did not affect that for [3H]alanine. Immune dot blot analysis using the antibody against L5 showed the presence of immunologically the same protein as L5 in the liver supernatant. Northern blot analysis of RNA in the immunoprecipitate prepared from the liver supernatant incubated with the antibody against L5 indicated that 5SrRNA was complexed with L5.(ABSTRACT TRUNCATED AT 250 WORDS)     

Amino acid selectivity in the aminoacylation of coenzyme A and RNA minihelices by aminoacyl-tRNA synthetases

Coenzyme A (CoA-SH), a cofactor in carboxyl group activation reactions, carries out a function in nonribosomal peptide synthesis that is analogous to the function of tRNA in ribosomal protein synthesis. The amino acid selectivity in the synthesis of aminoacyl-thioesters by nonribosomal peptide synthetases is relaxed, whereas the amino acid selectivity in the synthesis of aminoacyl-tRNA by aminoacyl-tRNA synthetases is restricted. Here I show that isoleucyl-tRNA synthetase aminoacylates CoA-SH with valine, leucine, threonine, alanine, and serine in addition to isoleucine. Valyl-tRNA synthetase catalyzes aminoacylations of CoA-SH with valine, threonine, alanine, serine, and isoleucine. Lysyl-tRNA synthetase aminoacylates CoA-SH with lysine, leucine, threonine, alanine, valine, and isoleucine. Thus, isoleucyl-, valyl-, and lysyl-tRNA synthetases behave as aminoacyl-S-CoA synthetases with relaxed amino acid selectivity. In contrast, RNA minihelices comprised of the acceptor-TpsiC helix of tRNA(Ile) or tRNA(Val) were aminoacylated by cognate synthetases selectively with isoleucine or valine, respectively. These and other data support a hypothesis that the present day aminoacyl-tRNA synthetases originated from ancestral forms that were involved in noncoded thioester-dependent peptide synthesis, functionally similar to the present day nonribosomal peptide synthetases.     

The biosynthesis of transfer RNA in insects. I. Increase of amino acid acceptor activity of specific tRNA's utilized for silk protein biosynthesis in the silk gland of Bombyx mori.

1) To detect the quantitative changes of amino acid acceptor activity of tRNA's from the posterior and middle silk glands of Bombyx mori at various ages, a relatively simple and rapid method was established using a mixture of radioactive amino acids in Chlorella hydrolysate. 2) The acceptor activities of silk gland tRNA for 15 amino acids tested seemed to be almost on the same level at the end of the 4th moult stage. During the 5th instar, however, characteristic increases were observed in glycine, alanine, and serine acceptor activities in both silk glands. 3) In the posterior silk gland, which produces fibroin, the acceptor activities for glycine and alanine increased more than that for serine. In the middle silk gland, which produces sericine, the acceptor activity for serine increased more than those for glycine and alanine. 4) In the light of observations on the increase of corresponding aminoacyl-tRNA synthetase activities in the silk glands, a functional adaptation of tRNA synthesis in the tissue is discussed.     

Initial position of aminoacylation of individual Escherichia coli, yeast, and calf liver transfer RNAs.

Transfer RNAs from Escherichia coli, yeast (Sacharomyces cerevisiae), and calf liver were subjected to controlled hydrolysis with venom exonuclease to remove 3'-terminal nucleotides, and then reconstructed successively with cytosine triphosphate (CTP) and 2'- or 3'-deoxyadenosine 5'-triphosphate in the presence of yeast CTP(ATP):tRNA nucleotidyltransferase. The modified tRNAs were purified by chromatography on DBAE-cellulose or acetylated DBAE-cellulose and then utilized in tRNA aminoacylation experiments in the presence of the homologous aminoacyl-tRNA synthetase activities. The E. coli, yeast, and calf liver aminoacyl-tRNA synthetases specific for alanine, glycine, histidine, lysine, serine, and threonine, as well as the E. coli and yeast prolyl-tRNA synthetases and the yeast glutaminyl-tRNA synthetase utilized only those homologous modified tRNAs terminating in 2'-deoxyadenosine (i.e., having an available 3'-OH group). This is interpreted as evidence that these aminoacyl-tRNA synthetases normally aminoacylate their unmodified cognate tRNAs on the 3'-OH group. The aminoacyl-tRNA synthetases from all three sources specific argining, isoleucine, leucine, phenylalanine, and valine, as well as the E. coli and yeast enzymes specific for methionine and the E. coli glutamyl-tRNA synthetase, used as substrates exclusively those tRNAs terminating in 3'-deoxyadenosine. Certain aminoacyl-tRNA synthetases, including the E. coli, yeast, and calf liver asparagine and tyrosine activating enzymes, the E. coli and yeast cysteinyl-tRNA synthetases, and the aspartyl-tRNA synthetase from yeast, utilized both isomeric tRNAs as substrates, although generally not at the same rate. While the calf liver aspartyl- and cysteinyl-tRNA synthetases utilized only the corresponding modified tRNA species terminating in 2'-deoxyadenosine, the use of a more concentrated enzyme preparation might well result in aminoacylation of the isomeric species. The one tRNA for which positional specificity does seem to have changed during evolution is tryptophan, whose E. coli aminoacyl-tRNA synthetase utilized predominantly the cognate tRNA terminating in 3'-deoxyadenosine, while the corresponding yeast and calf liver enzymes were found to utilize predominantly the isomeric tRNAs terminating in 2'-deoxyadenosine. The data presented indicate that while there is considerable diversity in the initial position of aminoacylation of individual tRNA isoacceptors derived from a single source, positional specificity has generally been conserved during the evolution from a prokaryotic to mammalian organism.     

Non-essential role of lysine residues for the catalytic activities of aspartyl-tRNA synthetase and comparison with other aminoacyl-tRNA synthetases

Essential lysine residues were sought in the catalytic site of baker's yeast aspartyl-tRNA synthetase (an alpha 2 dimer of Mr 125,000) using affinity labeling methods and periodate-oxidized adenosine, ATP, and tRNA(Asp). It is shown that the number of periodate-oxidized derivatives which can be bound to the synthetase via Schiff's base formation with epsilon-NH2 groups of lysine residues exceeds the stoichiometry of specific substrate binding. Furthermore, it is found that the enzymatic activities are not completely abolished, even for high incorporation levels of the modified substrates. The tRNA(Asp) aminoacylation reaction is more sensitive to labeling than is the ATP-PPi exchange one; for enzyme preparations modified with oxidized adenosine or ATP this activity remains unaltered. These results demonstrate the absence of a specific lysine residue directly involved in the catalytic activities of yeast aspartyl-tRNA synthetase. Comparative labeling experiments with oxidized ATP were run with several other aminoacyl-tRNA synthetases. Residual ATP-PPi exchange and tRNA aminoacylation activities measured in each case on the modified synthetases reveal different behaviors of these enzymes when compared to that of aspartyl-tRNA synthetase. When tested under identical experimental conditions, pure isoleucyl-, methionyl-, threonyl- and valyl-tRNA synthetases from E. coli can be completely inactivated for their catalytic activities; for E. coli alanyl-tRNA synthetase only the tRNA charging activity is affected, whereas yeast valyl-tRNA synthetase is only partly inactivated. The structural significance of these experiments and the occurrence of essential lysine residues in aminoacyl-tRNA synthetases are discussed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role for a conserved structural motif in assembly of a class I aminoacyl-tRNA synthetase active site

The catalytic domains of class I aminoacyl-tRNA synthetases are built around a conserved Rossmann nucleotide binding fold, with additional polypeptide domains responsible for tRNA binding or hydrolytic editing of misacylated substrates. Structural comparisons identified a conserved motif bridging the catalytic and anticodon binding domains of class Ia and Ib enzymes. This stem contact fold (SCF) has been proposed to globally orient each enzyme's cognate tRNA by interacting with the inner corner of the L-shaped tRNA. Despite the structural similarity of the SCF among class Ia/Ib enzymes, the sequence conservation is low. We replaced amino acids of the MetRS SCF with portions of the structurally similar glutaminyl-tRNA synthetase (GlnRS) motif or with alanine residues. Chimeric variants retained significant tRNA methionylation activity, indicating that structural integrity of the helix-turn-strand-helix motif contributes more to tRNA aminoacylation than does amino acid identity. In contrast, chimeras were significantly reduced in methionyl adenylate synthesis, suggesting a role for the SCF in formation of a structured active site domain. A highly conserved aspartic acid within the MetRS SCF is proposed to make an electrostatic interaction with an active site lysine; these residues were replaced with alanines or conservative substitutions. Both methionyl adenylate formation and methionine transfer were impaired, and activity was not significantly recovered by making the compensatory double substitution.     

Studies on chemical modification of thionucleosides in the transfer ribonucleic acid of Escherichia coli

(35)S-labelled tRNA from Escherichia coli was treated with chemical reagents such as CNBr, H(2)O(2), NH(2)OH, I(2), HNO(2), KMnO(4) and NaIO(4), under mild conditions where the four major bases were not affected. Gel filtration of the treated tRNA showed desulphurization to various extents, depending on the nature of the reagent. The treated samples after conversion into nucleosides were chromatographed on a phosphocellulose column. NH(2)OH, I(2) and NaIO(4) reacted with all the four thionucleosides of E. coli tRNA, 4-thiouridine (s(4)U), 5-methylaminomethyl-2-thiouridine (mnm(5)s(2)U), 2-thiocytidine (s(2)C) and 2-methylthio-N(6)-isopentenyladenosine (ms(2)i(6)A), to various extents. CNBr, HNO(2) and NaHSO(3) reacted with s(4)U, mnm(5)s(2)U and s(2)C, but not with ms(2)i(6)A. KMnO(4) and H(2)O(2) were also found to react extensively with thionucleosides in tRNA. Iodine oxidation of (35)S-labelled tRNA showed that only 6% of the sulphur was involved in disulphide formation. Desulphurization of E. coli tRNA with CNBr resulted in marked loss of acceptor activities for glutamic acid, glutamine and lysine. Acceptor activities for alanine, arginine, glycine, isoleucine, methionine, phenylalanine, serine, tyrosine and valine were also affected, but to a lesser extent. Five other amino acids tested were almost unaffected. These results indicate the fate of thionucleosides in tRNA when subjected to various chemical reactions and the involvement of sulphur in aminoacyl-tRNA synthetase recognition of some tRNA species of E. coli.     

Lysine 335, part of the KMSKS signature sequence, plays a crucial role in the amino acid activation catalysed by the methionyl-tRNA synthetase from Escherichia coli.

The KMSKS pattern, conserved among several aminoacyl-tRNA synthetase sequences, was first recognized in the Escherichia coli methionyl-tRNA synthetase through affinity labelling with an oxidized reactive derivative of tRNA(Met)f. Upon complex formation, two lysine residues of the methionyl-tRNA synthetase (Lys61 and 335, the latter being part of the KMSKS sequence) could be crosslinked by the 3'-acceptor end of the oxidized tRNA. Identification of an equivalent reactive lysine residue at the active centre of tyrosyl-tRNA synthetase designated the KMSKS sequence as a putative component of the active site of methionyl-tRNA synthetase. To probe the functional role of the labelled lysine residue within the KMSKS pattern, two variants of methionyl-tRNA synthetase containing a glutamine residue at either position 61 or 335 were constructed by using site-directed mutagenesis. Substitution of Lys61 slightly affected the enzyme activity. In contrast, the enzyme activities were very sensitive to the substitution of Lys335 by Gln. Pre-steady-state analysis of methionyladenylate synthesis demonstrated that this substitution rendered the enzyme unable to stabilize the transition state complex in the methionine activation reaction. A similar effect was obtained upon substituting Lys335 by an alanine instead of a glutamine residue, thereby excluding an effect specific for the glutamine side-chain. Furthermore, the importance of the basic character of Lys335 was investigated by studying mutants with a glutamate or an arginine residue at this position. It is concluded that the N-6-amino group of Lys335 plays a crucial role in the activation of methionine, mainly by stabilizing the transient complex on the way to methionyladenylate, through interaction with the pyrophosphate moiety of bound ATP-Mg2+. We propose, therefore, that the KMSKS pattern in the structure of an aminoacyl-tRNA synthetase sequence represents a signature sequence characteristic of both the pyrophosphate subsite and the catalytic centre.