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

摘要

Chloroplast NADP-malate dehydrogenase from Pisum sativum L. Giant leaves was purified to homogeneity and chemically modified with various reagents. The enzyme activity was considerably decreased by 2,3-butanedione, diethylpyrocarbonate, and iodoacetamide. The inactivation of the enzyme by 2,3-butanenedione or diethylpyrocarbonate was dependent on rime and reagent concentration. The reaction orders with respect to these reagents were 1.08 and 1.25, respectively. The inactivation by 1 mmol/L 2,3-butanedione was selectively and considerably protected by addition of 10 mmol/L oxaloacetate, to the extent of a 4-fold decrease of inactivation. The inactivation by diethylpyrocarbonate was selectively prevented by 10 mmol/L NADPH. The inactivated enzyme by diethylpyrocarbonate was almost completely restored by incubation with 20 mmol/L hydroxylamine. 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 histidyl residues are located at the active sire of the enzyme.