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

Previously we presented evidence for an essential arginine involved in binding inorganic pyrophosphate during catalysis by yeast inorganic pyrophosphatase [Cooperman, B. S., & Chiu, N. Y. (1973b) Biochemistry 12, 1676]. In the present work we show this residue to be arginine-77. Arginine-77 reacts with [14C]phenylgloxal considerably faster than the other five phenylglyoxal is selectively blocked in the presence of the competitive inhibitor calcium pyrophosphate. Our 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. In related work, we find the model compound N alpha-acetyl(diphenylglyoxal)arginine to hydrolyze 10 times more slowly at pH 4 than at pH 7. The high yields of derivatized peptides obtained in this work suggest the potential general utility of our procedure for locating arginine residues derivatized with phenylglyoxal within the covalent structure of proteins.