Presence of essential histidine residues in nadp-malic enzyme from maize

Abstract

Modification of maize leaf NADP-malic enzyme by diethylpyrocarbonate (DEP) caused rapid and complete inactivation of the enzyme. The inactivation followed pseudo-first-order reaction kinetics. The inactivation of the enzyme showed saturation kinetics with a half inactivation time, at saturating DEP, equal to 0.15 min and K DEP = 20 mM. The rate of inactivation was faster at 25° as compared to 0° ( t 0.5 0.75 min at 25° as against 5.6 min at 4° at 5 mM DEP). The enzyme was partially protected against DEP inactivation by NADP and complete protection was seen in the presence of NADP + Mg 2+ + malate or its analogues, thereby indicating that DEP modifies the active site. The modified enzyme showed an increase in absorbance at 240 nm which was lost upon treatment with 0.25 M NH 2OH and almost complete recovery of the enzyme activity was also observed. The results suggest that DEP modifies 3.0 residues per subunit and of these at least two residue per subunit can be modified without loss of activity in the presence of substrate. Modification of about one histidine residue is correlated with the loss of enzyme activity.