Affinity cleavage at the divalent metal site of porcine NAD-specific isocitrate dehydrogenase.
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 2alpha:1beta:1gamma. The results indicate that the oxidative inactivation and cleavage are specific and involve the 40 kDa alpha 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 alpha 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 alpha 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 alpha as the catalytic subunit of the multisubunit porcine NAD-dependent isocitrate dehydrogenase.
Huang
Y C
YC
Department of Chemistry and Biochemistry, University of Delaware, Newark 19716, USA.
Soundar
S
S
Colman
R F
RF
D000818:Animals; D001665:Binding Sites; D002413:Cations, Divalent / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000737:chemistry*; D007521:Isocitrate Dehydrogenase / Q000737:chemistry*; D007523:Isocitrates / Q000737:chemistry; D008024:Ligands; D008970:Molecular Weight; D009243:NAD / Q000737:chemistry*; D010446:Peptide Fragments / Q000737:chemistry; D011489:Protein Denaturation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013552:Swine
D002413:Cations, Divalent; D007523:Isocitrates; D008024:Ligands; D010446:Peptide Fragments; D009243:NAD; D007501:Iron; D007521:Isocitrate Dehydrogenase