Involvement of arginine residues in glutathione binding to yeast glyoxalase I.

Yeast glyoxalase I was inactivated by arginine-specific reagents. Inactivation by 2,3-butanedione, phenylglyoxal and camphorquinone 10-sulfonic acid followed pseudo first-order kinetics with the rate dependent upon modifier concentration. Extrapolation to complete inactivation showed modification of approx. two of the ten total arginyl residues in the native enzyme, with approx. one residue protected by glutathione (GSH) as determined by [ring-14C]phenylglyoxal incorporation. GSH protected the enzyme from inactivation, whereas methylglyoxal, glutathione disulfide (GSSG) and dithiothreitol afforded partial protection. The hemimercaptal of methylglyoxal and GSH and the catalytic product, S-lactoylglutathione provided substantial protection from inactivation. A methyl ester placed on the glycyl carboxyl moiety of GSH abolished all protective capability which suggests that this functionality is responsible for binding to the enzyme. These results provide the first evidence concerning the molecular binding mode of GSH to an enzyme. Arginyl residues are proposed as anionic recognition sites for glutathione on other GSH-utilizing enzymes.