Identification of an arginine important for enzymic activity within the covalent structure of yeast inorganic pyrophosphatase

Abstract

Evidence for an essential arginine involved in binding inorganic pyrophosphate during catalysis by baker''s yeast inorganic pyrophosphatase has been presented [Cooperman and Chiu]. This residue is shown to be arginine-77. Arginine-77 reacts with [14C]phenylglyoxal considerably faster than the other 5 arginine residues in the enzyme subunit, and its reaction with phenylglyoxal is selectively blocked in the presence of the competitive inhibitor calcium pyrophosphate. The procedure leading to the identification of Arg-77 utilizes the following steps: CNBr cleavage, digestion with Staphylococcus aureus V8 protease and with pepsin, and peptide mapping. All of these steps are performed below pH 5, a restriction imposed by the lability of the phenylglyoxal-arginine adduct at neutral pH. The model compound N.alpha.-acetyl(diphenylglyoxal)arginine hydrolyzed 10 times more slowly at pH 4 than at pH 7. The high yields of derivatized peptides obtained suggest the potential general utility of this procedure for locating arginine residues derivatized with phenylglyoxal within the covalent structure of proteins.