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.
Li
J
J
Department of Biochemistry, State University of New York, Buffalo 14214.
Pickart
C M
CM
D000217:Acyltransferases / Q000037:antagonists & inhibitors / Q000737:chemistry*; D000595:Amino Acid Sequence; D019881:Aminoacyltransferases; D001152:Arsenicals / Q000494:pharmacology*; D001483:Base Sequence; D001665:Binding Sites; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins; D012441:Saccharomyces cerevisiae / Q000201:enzymology
D001152:Arsenicals; D003432:Cross-Linking Reagents; D011994:Recombinant Proteins; C031672:4-((bromoacetyl)amino)phenyl arsenoxide; D000217:Acyltransferases; D019881:Aminoacyltransferases; C019727:arginyltransferase