ESSENTIAL ARGINYL AND HISTIDYL RESIDUES AT THE ACTIVE-SITE OF NADP-MALATE DEHYDROGENASE FROM PISUM-SATIVUM L GIANT LEAVES

Chloroplast NADP-malate dehydrogenase from Pisum sativum L. Giant leav es was purified to homogeneity and chemically modified with various re agents. The enzyme activity was considerably decreased by 2,3-butanedi one, diethylpyrocarbonate, and iodoacetamide. The inactivation of the enzyme by 2,3-butanenedione or diethylpyrocarbonate was dependent on r ime and reagent concentration. The reaction orders with respect to the se reagents were 1.08 and 1.25, respectively. The inactivation by 1 mm ol/L 2,3-butanedione was selectively and considerably protected by add ition of 10 mmol/L oxaloacetate, to the extent of a 4-fold decrease of inactivation. The inactivation by diethylpyrocarbonate was selectivel y prevented by 10 mmol/L NADPH. The inactivated enzyme by diethylpyroc arbonate was almost completely restored by incubation with 20 mmol/L h ydroxylamine. The reduced enzyme, rather than the oxidized enzyme, was more susceptible to the inactivation using diethylpyrocarbonate or 2, 3-butanedione. These results indicate that essential arginyl and histi dyl residues are located at the active sire of the enzyme.