Inactivation of pig heart NADP-specific isocitrate dehydrogenase by two affinity reagents is due to reaction with a cysteine not essential for function.
Pig heart NADP-dependent isocitrate dehydrogenase is 65% inactivated by 3-bromo-2-ketoglutarate (Ehrlich, R.S., and Colman, R.F., 1987, J. Biol. Chem. 262, 12,614-12,619) and 90% inactivated by 2-(4-bromo-2,3-dioxobutylthio)-1,N6- ethenoadenosine 2',5'-bisphosphate (2-BDB-T epsilon A-2',5'-DP) (Bailey, J.M., and Colman, R.F., 1987, J. Biol. Chem. 262, 12,620-12,626). Both inactivation reactions result in enzyme with an incorporation of 1.0 mol reagent/mol enzyme dimer and both modified enzymes bind only 1.0 mol manganous isocitrate or NADPH/mol enzyme dimer as compared to 2.0 mol manganous isocitrate or NADPH/mol enzyme dimer for unmodified enzyme. The inactivation reactions, which occur at or near the nucleotide binding site, are mutually exclusive. Reaction with either affinity reagent led to the isolation of the same modified triskaidekapeptide, DLAGXIHGLSNVK. We have isolated from isocitrate dehydrogenase a peptide, DLAGCIHGLSNVK, that had been modified by N-ethylmaleimide (NEM) with no loss of enzymatic activity. We now show that enzyme modified by NEM in the presence of isocitrate plus Mn2+ retains full catalytic activity but is not inactivated by either of the affinity reagents; thus, all three reagents appear to react at the same site. The analysis of HPLC tryptic maps of isocitrate dehydrogenase treated under denaturing conditions with iodo[3H]acetic acid or [3H]NEM demonstrates that both bromoketoglutarate and 2-BDB-T epsilon A-2',5'-DP react with the cysteine residue of DLAGCIHGLSNVK. We conclude that the cysteine of this triskaidekapeptide is close to the coenzyme binding site but is not essential for catalytic function.
Smyth
G E
GE
Department of Chemistry and Biochemistry, University of Delaware, Newark 19716.
Colman
R F
RF
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D000345:Affinity Labels / Q000494:pharmacology*; D000595:Amino Acid Sequence; D000818:Animals; D003545:Cysteine / Q000737:chemistry* / Q000187:drug effects / Q000502:physiology; D005033:Ethylmaleimide; D007521:Isocitrate Dehydrogenase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000502:physiology; D007656:Ketoglutaric Acids / Q000494:pharmacology*; D008929:Mitochondria, Heart / Q000201:enzymology* / Q000502:physiology; D008969:Molecular Sequence Data; D013552:Swine
D000345:Affinity Labels; D007656:Ketoglutaric Acids; D000244:Adenosine Diphosphate; C029384:3-bromo-2-ketoglutarate; C047570:2-((4-bromo-2,3-dioxobutyl)thio)-1,N(6)-ethenoadenosine 2',5'-bisphosphate; D007521:Isocitrate Dehydrogenase; C039355:isocitrate dehydrogenase (NADP+); D003545:Cysteine; D005033:Ethylmaleimide