Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:6.1.1.12 (aspartyl-tRNA synthetase)
 233 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Yeast phenylalanyl-tRNA synthetase (PRS) is shown to undergo autoaminoacylation with phenylalanine under in vitro amino acid activation conditions. Phenylalanyl adenylate enzyme complex yields a covalent phenylalanyl isopeptide exclusively with the beta subunit of the alpha 2 beta 2 enzyme. Contrary to previously reported cases of autoaminoacylation of aspartyl-tRNA synthetase and tryptophanyl-tRNA synthetase, the autoaminoacylation of PRS occurs under a specific set of conditions and results in the identification of only one labeled tryptic peptide on two types of high pressure liquid chromatography columns. The ability of PRS to undergo this covalent modification directly correlates with its ability to catalyze the synthesis of diadenosine 5',5"'-P1,P4-tetraphosphate from enzyme-bound phenylalanyl adenylate. Both reactions require the presence of low levels of zinc or cadmium and are inhibited by tRNAPhe or by low levels of low molecular weight thiols. Since diadenosine 5',5"'-P1,P4-tetraphosphate synthesis is known to be catalyzed in vivo in response to oxidation stress, it is also likely that the autoaminoacylation of phenylalanyl-tRNA synthetase may occur in vivo under a similar set of conditions. These reactions are thus not simply the result of accumulation of phenylalanyl adenylate and probably reflect conformational changes in the protein which are brought about by its interaction with zinc or cadmium.
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PMID:Covalent modification of phenylalanyl-tRNA synthetase with phenylalanine during the amino acid activation reaction catalyzed by the enzyme. 384 35

We describe a convenient, simple and novel continuous spectrophotometric method for the determination of aminoacyl-tRNA synthetase activity. The assay relies upon the measurement of inorganic pyrophosphate generated in the first step of the aminoacylation of a tRNA. Pyrophosphate release is coupled to inorganic pyrophosphatase, to generate phosphate, which in turn is used as the substrate of purine nucleoside phosphorylase to catalyze the N-glycosidic cleavage of 2-amino 6-mercapto 7-methylpurine ribonucleoside. Of the reaction products, ribose 1-phosphate and 2-amino 6-mercapto 7-methylpurine, the latter has a high absorbance at 360 nm relative to the nucleoside and hence provides a spectrophotometric signal that can be continuously followed. The non-destructive nature of the spectrophotometric assay allowed the re-use of the tRNAs in question in successive experiments. The usefulness of this method was demonstrated for glutaminyl-tRNA synthetase (GlnRS) and tryptophanyl-tRNA synthetase. Initial velocities measured using this assay correlate closely with those assayed by quantitation of [3H]Gln-tRNA or [14C]Trp-tRNA formation respectively. In both cases amino acid transfer from the aminoacyl adenylate to the tRNA represents the rate determining step. In addition, aminoacyl adenylate formation by aspartyl-tRNA synthetase was followed and provided a more sensitive means of active site titration than existing techniques. Finally, this novel method was used to provide direct evidence for the cooperativity of tRNA and ATP binding to GlnRS.
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PMID:A broadly applicable continuous spectrophotometric assay for measuring aminoacyl-tRNA synthetase activity. 765 11

In Escherichia coli, tyrosyl-tRNA synthetase is known to esterify tRNA(Tyr) with tyrosine. Resulting d-Tyr-tRNA(Tyr) can be hydrolyzed by a d-Tyr-tRNA(Tyr) deacylase. By monitoring E. coli growth in liquid medium, we systematically searched for other d-amino acids, the toxicity of which might be exacerbated by the inactivation of the gene encoding d-Tyr-tRNA(Tyr) deacylase. In addition to the already documented case of d-tyrosine, positive responses were obtained with d-tryptophan, d-aspartate, d-serine, and d-glutamine. In agreement with this observation, production of d-Asp-tRNA(Asp) and d-Trp-tRNA(Trp) by aspartyl-tRNA synthetase and tryptophanyl-tRNA synthetase, respectively, was established in vitro. Furthermore, the two d-aminoacylated tRNAs behaved as substrates of purified E. coli d-Tyr-tRNA(Tyr) deacylase. These results indicate that an unexpected high number of d-amino acids can impair the bacterium growth through the accumulation of d-aminoacyl-tRNA molecules and that d-Tyr-tRNA(Tyr) deacylase has a specificity broad enough to recycle any of these molecules. The same strategy of screening was applied using Saccharomyces cerevisiae, the tyrosyl-tRNA synthetase of which also produces d-Tyr-tRNA(Tyr), and which, like E. coli, possesses a d-Tyr-tRNA(Tyr) deacylase activity. In this case, inhibition of growth by the various 19 d-amino acids was followed on solid medium. Two isogenic strains containing or not the deacylase were compared. Toxic effects of d-tyrosine and d-leucine were reinforced upon deprivation of the deacylase. This observation suggests that, in yeast, at least two d-amino acids succeed in being transferred onto tRNAs and that, like in E. coli, the resulting two d-aminoacyl-tRNAs are substrates of a same d-aminoacyl-tRNA deacylase.
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PMID:Metabolism of D-aminoacyl-tRNAs in Escherichia coli and Saccharomyces cerevisiae cells. 1091 62