BINDING OF PHENYLARSENOXIDE TO ARG-TRANSFER-RNA PROTEIN TRANSFERASE IS INDEPENDENT OF VICINAL THIOLS

Reversible enzyme inhibition by phenylarsenoxides is generally taken t o indicate the presence of functionally important vicinal thiol groups , Arginyl aminoacyl-tRNA transferase from eukaryotes is potently inhib ited by phenylarsenoxides and possesses one or more essential sulfhydr yl groups [Li, J,, & Pickart, C. M, (1995) Biochemistry 34, 139-147]. To map the putative Cys residues that mediate arsenoxide binding to th e transferase from Saccharomyces cerevisiae, we systematically mutagen ized the 15 Cys residues of the transferase, singly and in combination , to Ala (13 Cys) or Ser (2 Cys), Six mutant enzymes, encompassing all 15 Cys residues of the transferase, were-characterized in detail. The results revealed that Cys-20, Cys-23, and Cys-94 and/or Cys-95 were i mportant for activity, since mutations at these positions reduced acti vity by 100-fold (Cys-94 and Cys-95 were mutated simultaneously). Surp risingly, however, all of the mutant enzymes retained the ability to b ind a radioiodinated phenylarsenoxide derivative, with undiminished st oichiometry and affinity. All of the mutant enzymes also remained susc eptible to irreversible reaction with a bifunctional phenylarsenoxide bearing a para-alkyl halide substituent, Prior reaction of the enzyme with the bifunctional reagent blocked subsequent binding of the radiol abeled phenylarsenoxide, indicating that these two reagents bind at a single common site. These results indicate that high-affinity binding of trivalent arsenicals can occur by a thiol-independent mechanism.