Identification of metal-isocitrate binding site of pig heart NADP-specific isocitrate dehydrogenase by affinity cleavage of the enzyme by Fe(2+)-isocitrate.
The divalent metal-isocitrate site of pig heart NADP-specific isocitrate dehydrogenase can be located by affinity cleavage of the enzyme by Fe(2+)-isocitrate in the presence of O2, in analogy to the "chemical nuclease" action of DNA-binding drugs linked to Fe-EDTA. The enzyme is irreversibly inactivated and cleaved by Fe(2+)-isocitrate more rapidly than by Fe2+. Mn2+ prevents inactivation and cleavage by Fe(2+)-isocitrate or by Fe2+. Furthermore, other tri- or dicarboxylates (such as citrate, tricarballylate, or malate), which are not effective substrates of the enzyme, fail to promote inactivation and cleavage of the enzyme by Fe2+. These results indicate that the oxidative inactivation and cleavage reactions are specific. Two pairs of major peptides are generated during Fe(2+)-isocitrate inactivation: 30 + 17 kDa and 35 + 11 kDa, as compared with 46 kDa for the intact enzyme. NH2-terminal sequencing revealed that these peptides arise by a mutually exclusive cleavage at either Asp253-Met254 or His309-Gly310, suggesting Asp253 and His309 as coordination sites for Fe(2+)-isocitrate and, by implication, for Mn(2+)-isocitrate. Fe2+ alone produces peptides (32 + 15 kDa) by an alternate specific cleavage between Tyr272 and Asp273, consistent with free metal ion occupying a different site from metal-isocitrate in NADP-dependent isocitrate dehydrogenase. Affinity cleavage may be a generally useful method for locating metal and metal-substrate sites in enzymes.
Soundar
S
S
Department of Chemistry and Biochemistry, University of Delaware, Newark 19716.
Colman
R F
RF
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D006868:Hydrolysis; D007501:Iron / Q000378:metabolism*; D007521:Isocitrate Dehydrogenase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D007523:Isocitrates / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D009206:Myocardium / Q000201:enzymology*; D010100:Oxygen / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D013552:Swine
D007523:Isocitrates; D010446:Peptide Fragments; D007501:Iron; D007521:Isocitrate Dehydrogenase; C039355:isocitrate dehydrogenase (NADP+); D010100:Oxygen