Aminoacyl-tRNA synthetases reaction mechanisms

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. 

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. 

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). 

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... 

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

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). 

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). 

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. 

The importance of the phosphoenzyme in the mechanism of action of succinyl-CoA synthetase in reactions (27a)-(27c) is also unknown. The mechanisms of action of aminoacyl-tRNA synthetases and of acyl-CoA synthetases do not include covalent enzymic intermediates. The fact that succinyl-CoA synthetase involves succinyl phosphate as the activated substrate, whereas the others involve acyl adenylates, does not explain the difference. There is no chemical catalytic basis for the mechanisms of the formation of these intermediates to vary in this way. Moreover, acetate kinase produces acetyl phosphate without the intermediate formation of a phosphoenzyme, so that at least acetate kinase has the capacity to catalyze direct phosphorylation of a carboxylate group. 

Figure 11.14 Amino acylation mechanisms catalyzed by aminoacyl-tRNA synthetases The two classes of aminoacyl-tRNA synthetases (aRS s) differ in the site of aminoacylation. Class I aRS s aminoacylate 2 -OH whereas class 11 aRS s add amino acids to 3 -OH of the terminal ribose of the 3 -terminal CCA of cognate tRNA. Magnesinm ions complexed with ATP to enter the active site of aRS may play a dual role in the activation step by both stabilizing the conformation of the ATP (Mg ion bridges the P- and y-phosphates) and participating in adenylate formation (second Mg is found between a- and P-phosphates in some aRS s). In class I aRS, both Lys of MSK and His of HIGH stabilize the bipyramidal oxyphosphorane transition state while R of motif 2 in class II aRS participates in the stabilization of the putative pentacoordinate transition state. The resulting mixed anhydride aminoacyl adenylate is held by the enzyme for the next reaction, i.e. the attack by the 2 -OH (class I) or 3 -OH (class II) of the terminal adenosine at the carbonyl of the aminoacyl adenylate. The amino acid then becomes esterified to the cognate tRNA.

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... 

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. 

The biosynthesis of a variety of biologically active peptides proceeds nucleic acid-free on protein templates (IK Peptide synthetases generally activate an acceptor amino acid by formation of amino-acyl adenylates or phosphates, which will be stabilized in an enzyne-aminoacylation step, similar as in tRNA-aminoacylation. Reaction with a donor peptide, which may be covalently bound, leads to a specific chain elongation. While small peptides like glutathione are formed by "one-step"-synthetases, more complex structures like gramicidin S are produced by multienzvme systems, which may contain multifunctional polypeptides. Characteristic features of such systems are 1.)activation as aminoacyl adenylates, 2.) aminoacylation of enzyme thiol-groups, 3.) covalently bound peptide intermediates and 4.) a specific intrinsic transport mechanism similar to the biosynthesis of fatty acids. 

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