Histidyl-Transfer-Ribonucleic-Acid Synthetase from <em>Salmonella typhimurium</em>.

The reactivity of the sulfhydryl groups of histidyl-tRNA 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 molecule of enzyme and the reaction is essentially 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 individuaily or in combination to the enzyme. The enzyme activity slowly changes after two or four sulfhydryl 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 K_m 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. [ABSTRACT FROM AUTHOR]