Affinity cleavage at the divalent metal site of porcine NAD‐specific isocitrate dehydrogenase

Publisher Website
Google Scholar
Abstract
A divalent metal ion, such as Mn2+, is required for the catalytic reaction and allosteric regulation of pig heart NAD‐dependent isocitrate dehydrogenase. The enzyme is irreversibly inactivated and cleaved by Fe2+ in the presence of O2 and ascorbate at pH 7.0. Mn2+ prevents both inactivation and cleavage. Nucleotide ligands, such as NAD, NADPH, and ADP, neither prevent nor promote inactivation or cleavage of the enzyme by Fe2+. The NAD‐specific isocitrate dehydrogenase is composed of three distinct subunits in the ratio 2α:1 β:1 γ. The results indicate that the oxidative inactivation and cleavage are specific and involve the 40 kDa α subunit of the enzyme. A pair of major peptides is generated during Fe2+ inactivation: 29.5 + 10.5 kDa, as determined by SDS‐PAGE. Amino‐terminal sequencing reveals that these peptides arise by cleavage of the Val262‐His263 bond of the α subunit. No fragments are produced when enzyme is incubated with Fe2+ and ascorbate under denaturing conditions in the presence of 6 M urea, indicating that the native structure is required for the specific cleavage. These results suggest that His263 of the α subunit may be a ligand of the divalent metal ion needed for the reaction catalyzed by isocitrate dehydrogenase. Isocitrate enhances the inactivation of enzyme caused by Fe2+ in the presence of oxygen, but prevents the cleavage, suggesting that inactivation occurs by a different mechanism when metal ion is bound to the enzyme in the presence of isocitrate: oxidation of cysteine may be responsible for the rapid inactivation in this case. Affinity cleavage caused by Fe2+ implicates α as the catalytic subunit of the multisubunit porcine NAD‐dependent isocitrate dehydrogenase.