title: Reactions of the sulfhydryl groups of alanyl-tRNA Synthetase.
abstract: The six sulfhydryl groups in each subunit of the alanyl-tRNA synthetase of Escherichia coli react with sulfhydryl reagents with at least four different rates. One reacts very rapidly with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), and a second reacts somewhat less rapidly with this reagent. These two groups are required for transfer activity, which is lost in proportion to the extent of derivatization. Two other groups react more slowly, with a consequent loss of exchange activity. The remaining two sulfhydryl groups do not react with DTNB until the protein is denatured. The inactivations are reversed by dithiothreitol. Two sulfhydryl groups react with N-ethylmaleimide (NEM) and with a spin-label derivative of NEM. These reactions resemble the modification of two sulfhydryl groups with DTNB, in that they also inactivate the transfer reaction but not the ATP:PPi exchange. The two spin labels are incorporated at similar rates but are in very different environments, one highly exposed and one highly immobilized. These groups do not interact with Mn2+, which is bound to the enzyme in the absence of ATP.
mesh_terms: D000412:Alanine-tRNA Ligase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D001665:Binding Sites; D055598:Chemical Phenomena; D002621:Chemistry; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004563:Electrochemistry; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000201:enzymology*; D005033:Ethylmaleimide / Q000494:pharmacology; D008345:Manganese / Q000378:metabolism; D013113:Spin Labels; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D013439:Sulfhydryl Reagents / Q000494:pharmacology
chemical_list: D013113:Spin Labels; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; D008345:Manganese; D004228:Dithionitrobenzoic Acid; D000604:Amino Acyl-tRNA Synthetases; D000412:Alanine-tRNA Ligase; D005033:Ethylmaleimide