The presence of functional arginine residues in phospho enolpyruvate carboxykinase from Saccharomyces cerevisiae

Abstract

Saccharomyces cerevisiae phospho enolpyruvate carboxykinase (ATP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.49) is completely inactivated by phenylglyoxal and 2,3-butanedione in borate buffer at pH 8.4, with pseudo-first-order kinetics and a second-order rate constant of 144 min −1 · M −1 and 21.6 min −1 · M −1, respectively. Phospho enolpyruvate, ADP and Mn 2+ (alone or in combination) protect the enzyme against inactivation, suggesting that the modification occurs at or near to the substrate-binding site. Almost complete restoration of activity was obtained when a sample of 2,3-butanedione-inactivated enzyme was freed of excess modifier and borate ions, suggesting that only arginyl groups are modified. The changes in the rate of inactivation in the presence of substrates and Mn 2+ were used to determine the dissociation constants for enzyme-ligand complexes, and values of 23 ± 3 μM, 168 ±44 μM and 244 ± 54 μM were found for the dissociation constants for the enzyme-Mn 2+, enzyme-ADP and enzyme-phospho enolpyruvate complexes, respectively. Based on kinetic data, it is shown that 1 mol of reagent must combine per enzyme active unit in order to inactivate the enzyme. Complete inactivation of the carboxykinase can be correlated with the incorporation of 3–4 mol [7- 14C]phenylglyoxal per mol of enzyme subunit. Assuming a stoichiometry of 1:1 between phenylglyoxal incorporation and arginine modification, our results suggest that the modification of only two of the three to four reactive arginine residues per phospho enolpyruvate carboxykinase subunit is responsible for inactivation.