Inactivation of arginyl-tRNA protein transferase by a bifunctional arsenoxide: identification of residues proximal to the arsenoxide site.

Aminoacyl-tRNA protein transferases catalyze (posttranslational) aminoacylation of specific protein N-termini, using aminoacyl-tRNA as substrate. This modification targets the protein for ATP-dependent degradation; in eukaryotes, degradation occurs in the ubiquitin-mediated pathway. The eukaryotic transferase, which catalyzes Arg transfer to N-terminal Glu or Asp residues, is potently inhibited by phenylarsenoxides. The gene encoding Arg-tRNA protein transferase from the yeast Saccharomyces cerevisiae was subcloned and overexpressed in Escherichia coli to provide large amounts of homogeneous protein for a molecular analysis of this inhibition. The bifunctional reagent para-[(bromoacetyl)amino]-phenylarsenoxide is a potent and irreversible inactivator of the yeast transferase; the arsenoxide moiety of the reagent directs binding to the enzyme, while the alkyl halide moiety alkylates a residue(s) proximal to the arsenoxide site. One mole of 14C-labeled reagent was covalently incorporated during inactivation, with the side chain of Cys-315 representing the major site of alkylation. Mutation of Cys-315 to Ala yielded a fully active enzyme which was still subject to stoichiometric, irreversible inactivation by the bifunctional arsenoxide. With the C315A-enzyme, the major fraction of the 14C-labeled bifunctional reagent was associated with the side chain(s) of one or more of a stretch of Glu residues (Glu 339-341). These results show that phenylarsenoxides inhibit Arg-tRNA protein transferase by binding to a site that is either itself essential, or regulates an essential site. Inhibition appears to occur through a steric blockade mechanism.