Aminoacyl-tRNA synthetases aminoacylation mechanisms

The attachment of an amino acid to a tRNA is catalyzed by an enzyme called aminoacyl-tRNA synthetase. The mechanism for the reaction is shown here. 

One important question is that of the order in which the basic mechanisms of evolution processes, leading eventually to the emergence of life, occurred. As far as the development of the genetic code is concerned, it is not clear whether the code evolved prior to the aminoacylation process, i.e., whether aminoacyl-tRNA synthetases evolved before or after the code. A tRNA species which is aminoacy-lated by two different synthetases was studied if this tRNA had important identity elements such as the discriminator base and the three anticodon bases for the two synthetases, this would be evidence that the aminoacyl-tRNA synthetases had developed after the genetic code. Dieter Soil s group, which is experienced in working with this family of enzymes, came to the conclusion that the universal genetic code must have developed before the evolution of the aminoacylation system (Hohn et al, 2006). 

For most amino acids, the ester linkage between the ct-COOH group of the amino acid and the 3 -terminal adenosine of a cognate tRNA is formed in a two-step mechanism catalyzed by an aminoacyl-tRNA synthetase (aaRS). ° In this so-called direct pathway, the aaRS first catalyzes the reaction of the amino acid with adenosine triphosphate (ATP), yielding the enzyme-bound high-energy intermediate aa AMP and PPi in the second step, this aaRS-bound intermediate reacts with tRNA to yield aa-tRNA and AMP (Figure 1). 

R. Chenevert S. Bernier J. Lapointe, Inhibitors of Aminoacyl-tRNA Synthetases as Antibiotics and Tools for Structural and Mechanistic Studies. In Translation Mechanisms J. Lapointe, L. Brakier-Gingras, Eds. Eurekah.com/Landes Bioscience and Kluwer Academic/Plenum Publishers Georgetown TX, 2003 pp 416-428. 

Figure 20.25 Regulation of the activities of the aminoacyl-tRNA synthetases by the concentrations of free tRNAs (i.e. uncharged tRNA). Changes in the concentrations of free tRNAs provide the mechanism for communication between control via the initiation factor (Figure 20.20) and ribosomal protein kinase (steps 6 and 7) and the flux-generating step.

The structures of all the aminoacyl-tRNA synthetases of E. coli have been determined. Researchers have divided them into two classes (Table 27-7) based on substantial differences in primary and tertiary structure and in reaction mechanism (Fig. 27-14) these two classes are the same in all organisms. There is no evidence for a common ancestor, and the biological, chemical, or evolutionary reasons for two enzyme classes for essentially identical processes remain obscure. 

MECHANISM FIGURE 27-14 Aminoacylation of tRNA by aminoacyl-tRNA synthetases. Step is formation of an aminoacyl adenylate, which remains bound to the active site. In the second step the aminoacyl group is transferred to the tRNA. The mechanism of this step is somewhat different for the two classes of aminoacyl-tRNA synthetases (see Table 27-7). For class I enzymes, (2a) the aminoacyl group is transferred initially to the 2 -hydroxyl group of the 3 -terminal A residue, then (3a) to the 3 -hydroxyl group by a transesterification reaction. For class II enzymes, ( the... 

While peptide antibiotics are synthesized according to enzyme-controlled polymerization patterns, both proteins and nucleic acids are made by template mechanisms. Tire sequence of their monomer emits is determined by genetically encoded information. A key reaction in the formation of proteins is the transfer of activated aminoacyl groups to molecules of tRNA (Eq. 17-36). Tire tRNAs act as carriers or adapters as explained in detail in Chapter 29. Each aminoacyl-tRNA synthetase must recognize the correct tRNA and attach the correct amino acid to it. The tRNA then carries the activated amino acid to a ribosome, where it is placed, at the correct moment, in the active site. Peptidyltransferase, using a transacylation reaction, in an insertion mechanism transfers the C terminus of the growing peptide chain onto the amino group of... 

Mechanisms of reaction. Activation of an amino acid occurs by a direct in-line nucleophilic displacement by a carboxylate oxygen atom of the amino acid on the a phosphorus atom of MgATP to form the aminoacyl adenylate (Eq. 29-1, step a). For yeast phenylalanyl-tRNA synthetases the preferred form of MgATP appears to be the P,y-bidentate (A screw sense) complex (p. 643).250 This is followed by a second nucleophilic displacement, this one on the C = 0 group of the aminoacyl adenylate by the -OH group of the tRNA (Eq. 29-1, step b Fig. 29-9C). A conformational change in the protein may be required to permit dissociation of the product, the aminoacyl-tRNA. In the complex of a class I synthetase with aminoacyl... 

Figure 13.4 The double sieve analogy for the editing mechanism of the isoleucyl-tRNA synthetase. The active site for the formation of the aminoacyl adenylate can exclude amino acids that are larger than isoleucine but not those that are smaller. On the other hand, a hydrolytic site that is just large enough to bind valine can exclude isoleucine while accepting valine and all the smaller amino acids. (In some enzymes, the hydrolytic site offers specific chemical interactions that enable it to bind isosteres of the correct amino acid as well as smaller amino acids.)...

J. J. Hopfield has suggested a general mechanism called kinetic proofreading in which there is no hydrolytic site on the enzyme instead, the desired intermediates diffuse into solution, where they hydrolyze nonenzymatically.54 An example is in the selection of amino acids by the aminoacyl-tRNA synthetases (equation 13.32). 

Example. A detailed mechanism for one of the reactions catalyzed by aminoacyl-tRNA-synthetase [59] is represented by the set of steps... 

Aminoacyl-tRNA synthetases (aaRSs) are critical components of the translation machinery for protein synthesis in every living cell (1). Each aaRS enzyme in this family links a single amino acid covalently to one or more tRNA isoacceptors to form charged tRNAs. Identity elements within the tRNAs serve as molecular determinants or antideterminants that aid in selection by cognate aaRSs (2). Some aaRSs also have an amino acid editing mechanism to clear their mistakes (3). The canonical aaRSs and aaRS-like proteins have functionally diverged to perform many other important roles in the cell (4, 5). Their versatility and adaptability have provided unique opportunities to develop biotechnology tools and to advance medical research. 

Both classes of enzymes catalyze the common aminoacylation reaction but via different mechanisms (1). Class I and Class II aaRSs bind ATP in an extended and bent conformation, respectively (Fig. 2). In addition, class I enzymes bind the tRNA acceptor stem from the minor groove side, which orients the 2 -hydroxyl group of the A76 ribose for attachment of the amino acid (Fig. 3). In contrast. Class II aaRSs aminoacylate the 3 -hydroxyl of the terminal adenosine, because the enzyme binds to tRNA via its major groove. Class II phenylalanyl-tRNA synthetase (PheRS), which charges amino acids onto the 2 -hydroxyl group of A76 of tRNA , is the only known exception to this rule. 

Hendrickson T, Schimmel P. Transfer RNA-dependent amino acid discrimination by aminoacyl-tRNA synthetases. In Translation Mechanisms. Lapointe J, Brakier-Gingras L, eds. 2003. Kluwer Academic/Plenum Publishers, New York. p. 34-64. 

In several bacterial species, uncharged tRNA serves as the effector molecule in controlling expression of several aminoacyl-tRNA synthetase genes and a few amino acid biosynthetic oper-ons by a conunon mechanism termed T-box antitermination. 

Comprehensive Biological Catalysis—a Mechanistic Reference Volume has recently been published. The fiiU contents list (approximate number of references in parentheses) is as follows S-adenosylmethionine-dependent methyltransferases (110) prenyl transfer and the enzymes of terpenoid and steroid biosynthesis (330) glycosyl transfer (800) mechanism of folate-requiring enzymes in one-carbon metabohsm (260) hydride and alkyl group shifts in the reactions of aldehydes and ketones (150) phosphoenolpyruvate as an electrophile carboxyvinyl transfer reactions (140) physical organic chemistry of acyl transfer reactions (220) catalytic mechanisms of the aspartic proteinases (90) the serine proteinases (135) cysteine proteinases (350) zinc proteinases (200) esterases and lipases (160) reactions of carbon at the carbon dioxide level of oxidation (390) transfer of the POj group (230) phosphate diesterases and triesterases (160) ribozymes (70) catalysis of tRNA aminoacylation by class I and class II aminoacyl-tRNA synthetases (220) thio-disulfide exchange of divalent sulfirr (150) and sulfotransferases (50). 

C.W. Carter Jr. 1993. Cognihon, mechanism, and evolutionary relahonships in aminoacyl-tRNA synthetases Rev. Biochem. 62 715-748. (PubMed)... 

Summary Stimulating effects of isopropoxygermatran (IPG) and I-etoxysilatran (ES) on liver recovery occur through activation of protein-synthesizing components, aminoacyl-tRNA-synthetases (ARSes) in particular. Increase in the activity of preparations of total ARSes has been shown. Several aspects of the mechanisms of stimulating effects of MA were discussed. 

Although the accuracy of translation (approximately one error per 104 amino acids incorporated) is lower than those of DNA replication and transcription, it is remarkably higher than one would expect of such a complex process. The principal reasons for the accuracy with which amino acids are incorporated into polypeptides include codon-anticodon base pairing and the mechanism by which amino acids are attached to their cognate tRNAs. The attachment of amino acids to tRNAs, considered the first step in protein synthesis, is catalyzed by a group of enzymes called the aminoacyl-tRNA synthetases. The precision with which these enzymes esterify each specific amino acid to the correct tRNA is now believed to be so important for accurate translation that their functioning has been referred to collectively as the second genetic code. 

The best investigated mechanism, distributed ubiquitously in Hving matter, is the template-dependent ribosomal synthesis of proteins. Here the amino acids are activated by adenylation catalyzed by aminoacyl-tRNA synthetases [1]. 

In the activation of amino acids for protein biosynthesis, the aminoacyl-tRNA synthetases catalyze their ligation as acyl esters to the 3-hydroxyl ends of their cognate species of tRNA. The chemical activation mechanism requires ATP and occurs in two steps, the activation of the amino acid by reaction with ATP to form an aminoacyl adenylate in reaction (29a), and the transfer of the activated aminoacyl group to the 3 -hydroxyl end of tRNA in reaction (29b) (88). 

The nucleotidyl transfer step is reaction (29a), which proceeds with inversion of configuration at phosphorus in all of the aminoacyl-tRNA synthetase reactions so far studied [for amino acids (aa) Phe, He, Tyr, and Met] (89-92). Stereochemical inversion shows that the nucleotidyl transfer mechanism involves an uneven number of substitutions on phosphorus. Since no other evidence of an adenylyl-enzyme can be found, aminoacyl activation most likely occurs by a single-displacement mechanism, with direct transfer of the AMP group from ATP to the carboxylate group of the amino acid within the enzyme-amino acid-ATP complex. 

Acetyl-CoA synthetase from mammalian tissues and yeast catalyzes the reaction of acetate with ATP and CoA to form acetyl-CoA by a chemical mechanism similar to that of the aminoacyl-tRNA synthetases. The catalytic pathway is similar to that of reactions (29a) and (29b), substituting acetate for the amino acid and CoA for tRNA (93). The activation of acetate via the intermediate acetyl adenylate also occurs with inversion of configuration at P of ATP (94). Thus, as for aminoacyl-tRNA synthetases, acetyl-CoA synthetase appears to catalyze the activation of acetate by a single-displacement mechanism. 

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