Characterization of an active site peptide modified by the substrate analogue 3-bromo-2-ketoglutarate on a single chain of dimeric NADP+-dependent isocitrate dehydrogenase.
The substrate analogue 3-bromo-2-ketoglutarate reacts with pig heart NADP+-dependent isocitrate dehydrogenase to yield partially inactive enzyme. Following 65% inactivation, no further inactivation was observed. Concomitant with this inactivation, incorporation of 1 mol of reagent/mol of enzyme dimer was measured. The dependence of the inactivation rate on bromoketoglutarate concentration is consistent with reversible binding of reagent (KI = 360 microM) prior to irreversible reaction. Manganous isocitrate reduces the rate of inactivation by 80% but does not provide complete protection even at saturating concentrations. Complete protection is obtained with NADP+ or the NADP+-alpha-ketoglutarate adduct. By modification with [14C]bromoketoglutarate or by NaB3H4 reduction of modified enzyme, a single major radiolabeled tryptic peptide was obtained by high performance liquid chromatography with the sequence: Asp-Leu-Ala-Gly-X-Ile-His-Gly-Leu-Ser-Asn-Val-Lys. Evidence in the following paper (Bailey, J.M., Colman, R.F. (1987) J. Biol. Chem. 262, 12620-12626) indicates that X is glutamic acid. Enzyme modified at the coenzyme site by 2-(bromo-2,3-dioxobutylthio)-1,N(6)-ethenoadenosine 2',5'-biphosphate in the presence of manganous isocitrate is not further inactivated by bromoketoglutarate. Bromoketoglutarate-modified enzyme exhibits a stoichiometry of binding isocitrate and NADPH equal to 1 mol/mol of enzyme dimer, half that of native enzyme. These results indicate that bromoketoglutarate modifies a residue in the nicotinamide region of the coenzyme site proximal to the substrate site and that reaction at one catalytic site of the enzyme dimer decreases the activity of the other site.
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D007521:Isocitrate Dehydrogenase / Q000378:metabolism*; D007523:Isocitrates / Q000494:pharmacology; D007656:Ketoglutaric Acids / Q000494:pharmacology*; D007700:Kinetics; D008345:Manganese / Q000494:pharmacology; D017895:Manganese Compounds; D009249:NADP / Q000494:pharmacology; D010446:Peptide Fragments / Q000032:analysis; D013431:Sulfates / Q000494:pharmacology; D013552:Swine
D007523:Isocitrates; D007656:Ketoglutaric Acids; D017895:Manganese Compounds; D010446:Peptide Fragments; D013431:Sulfates; D008345:Manganese; D009249:NADP; C029384:3-bromo-2-ketoglutarate; C034219:isocitric acid; D007521:Isocitrate Dehydrogenase; C039355:isocitrate dehydrogenase (NADP+); C039798:manganese sulfate