INACTIVATION OF ARGINYL-TRANSFER-RNA PROTEIN TRANSFERASE BY A BIFUNCTIONAL ARSENOXIDE - IDENTIFICATION OF RESIDUES PROXIMAL TO THE ARSENOXIDE SITE

Aminoacyl-tRNA protein transferases catalyze (posttranslational) amino acylation of specific protein N-termini, using aminoacyl-tRNA as subst rate. This modification targets the protein for ATP-dependent degradat ion; in eukaryotes, degradation occurs in the ubiquitin-mediated pathw ay. The eukaryotic transferase, which catalyzes Arg transfer to N-term inal Glu or Asp residues, is potently inhibited by phenylarsenoxides. The gene encoding Arg-tRNA protein transferase from the yeast Saccharo myces cerevisiae was subcloned and overexpressed in Escherichia coli t o provide large amounts of homogeneous protein for a molecular analysi s of this inhibition. The bifunctional reagent para- [(bromoacetyl)ami no]phenylarsenoxide is a potent and irreversible inactivator of the ye ast transferase; the arsenoxide moiety of the reagent directs binding to the enzyme, while the alkyl halide moiety alkylates a residue(s) pr oximal to the arsenoxide site. One mole of C-14-labeled reagent was co valently 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 stoichiom etric, irreversible inactivation by the bifunctional arsenoxide. With the C315A-enzyme, the major fraction of the C-14-labeled bifunctional reagent was associated with the side chain(s) of one or more of a stre tch of Glu residues (Glu 339-341). These results show that phenylarsen oxides 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.