Chemical modifications of Bacillus subtilis tryptophanyl-tRNA synthetase.
A concerted conformational change in Bacillus subtilis tryptophanyl-tRNA synthetase (TrpRS) was evident from previous fluorescence on the quenching of the single Trp residue Trp-92 in the 4FTrp-AMP complexed enzyme. In this study, chemical modifications of the B. subtilis TrpRS were employed to further characterize this conformational change, with the single Trp residue serving as a marker for monitoring the change. Modifications of the enzyme by means of the Trp-specific agent N-bromosuccinimide (NBS) or 3-bromo-3-methyl-2-(2-nitrophenylmercapto)-3H-indole (BNPS-skatole) inactivated the enzyme in accord with the essential role of Trp-92, as identified previously by site-directed mutagenesis. ATP sensitized TrpRS toward inactivation by NBS and BNPS-skatole, which suggested a conformational change that resulted in greater accessibility of Trp-92 toward modifications. In contrast, the cognate tRNATrp substrate exerted a specific protective effect against inactivation by both of the reagents, indicating that the TrpRS-tRNATrp interaction reduces the accessibility of Trp-92 under our experimental conditions. By comparison, modification of sulfhydryl groups by means of iodoacetamide did not reduce TrpRS activity. Observations on Trp-specific modification and substrate protection effects are discussed in the context of the Bacillus stearothermophilus TrpRS crystal structure.
Xue
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Department of Biochemistry, Hong Kong University of Science and Technology, Clear Water Bay. hxue@usthk.ust.hk
Xue
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Doublié
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Carter
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CW
D001412:Bacillus subtilis / Q000201:enzymology*; D001665:Binding Sites / Q000187:drug effects; D001974:Bromosuccinimide / Q000494:pharmacology; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D007460:Iodoacetamide / Q000494:pharmacology; D011487:Protein Conformation / Q000187:drug effects; D012862:Skatole / Q000031:analogs & derivatives / Q000494:pharmacology; D013438:Sulfhydryl Compounds / Q000378:metabolism; D014364:Tryptophan / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D014369:Tryptophan-tRNA Ligase / Q000737:chemistry* / Q000187:drug effects / Q000302:isolation & purification
D013438:Sulfhydryl Compounds; C005904:BNPS-skatole; D014364:Tryptophan; D012862:Skatole; D014369:Tryptophan-tRNA Ligase; D003545:Cysteine; D001974:Bromosuccinimide; D007460:Iodoacetamide