The presence of essential histidine residues in phospho enolpyruvate carboxylase from maize leaves

Abstract

Diethylpyrocarbonate inactivated maize leaf phospho enolpyruvate carboxylase (EC 4.1.1.31) with a second-order rate constant of 6.9 mM −1·min −1. Activity could be restored by treatment with hydroxylamine and the pH curve of inactivation suggested the involvement of a residue having a p K a of 6.9. The ultraviolet difference spectrum of modified versus native enzyme revealed the formation of ethoxyformyl histidine residues. Phospho enolpyruvate, plus or minus MgCl 2, protected the enzyme against inactivation, suggesting that the modification occurred at or near the substrate-binding site. Under denaturating conditions, seven histidine residues per 100-kDa subunit were modified by diethylpyrocarbonate. Under non-denaturating conditions, the carboxylase lost all of its activity after about four of the seven histidine residues per subunit had been modified by the reagent. In experiment using phospho enolpyruvate, plus or minus MgCl 2, as protective agent, it was observed that complete inactivation resulted from the modification of only two histidine residues per subunit. On the basis of these results it is suggested that two of the four accessible histidines in the native enzyme subunit are essential for substrate binding or catalysis by maize leaf phospho enolpyruvate carboxylase.