Histidyl-transfer-ribonucleic-acid synthetase from Salmonella typhimurium. Studies of the sulfhydryl groups.
The reactivity of the sulfhydryl groups of histidyl-t RNA synthetase from Salmonella typhimurium and the effect of substrates on the reactivity has been studied using p-hydroxymercuribenzoate and 5, 5'-dithiobis (2-nitrobenzoic acid) as reagents. It has been found that 5, 5'-dithiobis (2-nitrobenzoic acid) titrates only two sulfhydryl groups per molcule of enzyme and the reaction is essenaitlly monophasic, while p-hydroxymercuribenzoate titrates four sulhydryl groups. As observed kinetically the reaction with p-hydroxymercuribenzoate is strongly biphasic, each phase corresponding to about two sulfhydryl groups per enzyme molecule. With both reagents no detectable difference in sulfhydryl group reactivity was observed when ATP, histidine and tRNA specific for histidine were added individually or in combination to the enzyme. The enzyme activity slowly changes after two or four sulhydryl groups are blocked by 5, 5'-dithiobis (2-nitrobenzoic acid) or p-hydroxymercuribenzoate respectively. A new, stable level of activity is reached that is characterized by a different Km value for the aminoacylation reaction. The results indicate that the sulfhydryl groups reacting with the two reagents used here are neither directly involved in the binding of the substrates nor in the catalytic process. The ultimate change in enzyme activity after reaction of the sulfhydryl groups suggests a transition to an alternative enzyme structure.
D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism; D001665:Binding Sites; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D006643:Histidine-tRNA Ligase / Q000378:metabolism; D006902:Hydroxymercuribenzoates / Q000494:pharmacology; D007700:Kinetics; D011485:Protein Binding; D011487:Protein Conformation; D012486:Salmonella typhimurium / Q000201:enzymology*; D013438:Sulfhydryl Compounds / Q000032:analysis
D006902:Hydroxymercuribenzoates; D013438:Sulfhydryl Compounds; D004228:Dithionitrobenzoic Acid; D000604:Amino Acyl-tRNA Synthetases; D006643:Histidine-tRNA Ligase