Binding of phenylarsenoxide to Arg-tRNA protein transferase is independent of vicinal thiols.
Reversible enzyme inhibition by phenylarsenoxides is generally taken to indicate the presence of functionally important vicinal thiol groups. Arginyl aminoacyl-tRNA transferase from eukaryotes is potently inhibited by phenylarsenoxides and possesses one or more essential sulfhydryl groups (Li, J., & Pickart C. M. (1995) Biochemistry 34, 139-147]. To map the putative Cys residues that mediate arsenoxide binding to the transferase from Saccharomyces cerevisiae, we systematically mutagenized 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 important for activity, since mutations at these positions reduced activity by 100-fold (Cys-94 and Cys-95 were mutated simultaneously). Surprisingly, however, all of the mutant enzymes retained the ability to bind a radioiodinated phenylarsenoxide derivative, with undiminished stoichiometry and affinity. All of the mutant enzymes also remained susceptible to irreversible reaction with a bifunctional phenylarsenoxide bearing a paraalkyl halide substituent. Prior reaction of the enzyme with the bifunctional reagent blocked subsequent binding of the radiolabeled 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.
D000217:Acyltransferases / Q000235:genetics / Q000378:metabolism*; D000409:Alanine / Q000235:genetics; D019881:Aminoacyltransferases; D001152:Arsenicals / Q000378:metabolism*; D001483:Base Sequence; D003545:Cysteine / Q000235:genetics; D017931:DNA Primers; D007457:Iodine Radioisotopes; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D012694:Serine / Q000235:genetics; D013438:Sulfhydryl Compounds / Q000378:metabolism*
D001152:Arsenicals; D017931:DNA Primers; D007457:Iodine Radioisotopes; D013438:Sulfhydryl Compounds; C029341:oxophenylarsine; D012694:Serine; D000217:Acyltransferases; D019881:Aminoacyltransferases; C019727:arginyltransferase; D003545:Cysteine; D000409:Alanine