The reaction of isoleucyl-tRNA synthetase (IRS) with N-ethylmaleimide (NEM) and p-hydroxymercuribenzoate (pMB) has been used to probe the role of sulfhydryl groups in the catalytic and tRNA recognition activities of the enzyme. The reaction of the protein with NEM has revealed one rapidly reacting sulfhydryl group and a class that reacts less than 10% as fast. Modification of the fast-reacting group with NEM markedly reduces the ATP-PPi exchange activity and the formation of Ile-tRNA but has no detectable effect on either the equilibrium constant or the rate of binding of tRNAIle to the enzyme. In the presence of isoleucine and ATP, the reaction with NEM and the attendant loss of activities are prevented; other amino acids, other triphosphates, or tRNA, AMP and PP, do not protect the enzyme. The modification produced by reaction with NEM decreases the rate but not the extent of IRS(Ile-AMP) complex formation; thus, each of the altered molecules can react with isoleucine and ATP to form the enzyme-bound Ile-AMP but at less than 1% the rate. With native IRS, binding of isoleucine stimulates the rate of association and dissociation of tRNAIle from the enzyme (Yarus & Berg, 1969); with NEM-modified IRS, this effect of isoleucine is reduced, indicating that the modification has interrupted the linkage between the isoleucine catalytic site and the tRNA binding site. Nine sulfhydryl groups per mole of IRS are revealed by titration with pMB. Inactivation of the ATP-PPi exchange reaches a limiting value of 90% when all of the sulfhydryl groups are titrated: Ile-tRNA synthesis, however, is inhibited more than 99% when five sulfhydryl groups are titrated. In contrast to the NEM-modification, binding of the mercurial to IRS markedly decreases the capacity of the protein to bind its cognate tRNA. The data suggest that there is one sulfhydryl group at or near the catalytic site, which must be free for maximal rate of Ile-AMP formation and utilization: this sulfhydryl group has no role in tRNA binding. Other sulfhydryl groups may contribute to the specific binding of the cognate tRNA either directly or by maintaining the needed protein conformation. © 1969.
