Inactivation of phospho enolpyruvate carboxylase from maize leaves by modification with phenylglyoxal

Abstract

Phenylglyoxal rapidly and completely inactivated maize leaf phospho enolpyruvate carboxylase. Inactivation exhibited pseudo-first-order kinetics with a reaction order of approximately one and a second-order rate constant of 8.32 mM −1 · min −1. Phospho enolpyruvate, and more effectively phospho enolpyruvate plus MgCl 2, protected the enzyme against inactivation, suggesting that the modification occurs at or near to the substrate-binding site. Malate and oxalacetate also prevented inactivation. The protective effects of phospho enolpyruvate (in the absence or in the presence of MgCl 2) and malate were studied at pH 7.0, 7.5 and 7.9. In the absence of MgCl 2, phospho enolpyruvate was more effective at pH 7.5, while in the presence of MgCl 2 the protection increased along with the pH. On the other hand, in the absence of MgCl 2, malate at pH 7 afforded higher protection than phospho enolpyruvate. Loss of enzymic activity was directly proportional to the incorporation of [2- 14C]phenylglyoxal at least until 30% of the initial activity remained. Complete inactivation of the carboxylase was correlated with the incorporation of 8 mol [2- 14C]phenylglyoxal per mol enzyme. When phospho enolpyruvate plus MgCl 2 were used as protective agents, this number reduced to 4. Assuming a stoichiometry of 2:1 between phenylglyoxal incorporation and arginine modification, these results suggest that only two arginine residues per phospho enolpyruvate carboxylase molecule are responsible for inactivation.