On the mechanism of NADP+-linked isocitrate dehydrogenase from heart mitochondria. II. The transient-state kinetics of the oxidative decarboxylation of isocitrate.
The kinetics of a single turnover of enzyme-catalysed oxidative decarboxylation have been studied by mixing a stoichiometric complex of enzyme, isocitrate and Mg2+ with large concentrations of NADP+ in a stopped-flow apparatus, and monitoring the formation of NADPH by fluorescence measurements. A transient is revealed that exhibits enhanced nucleotide fluorescence and is not detectable by light absorption measurements. The results obtained with the largest NADP+ concentrations, such that the product NADPH is largely displaced from its enzyme complex, show that a step that precedes the release of free NADPH is rate-limiting in the oxidative decarboxylation reaction under conditions of catalytic cycling. The rate constants for this step, tentatively identified as the formation of the complex of enzyme, Mg2+ and NADPH from a precursor NADPH-containing complex, and for the dissociation of NADPH from this complex have been estimated from the integrated rate equation for a simple model for the product phase of the reaction, by methods of nonlinear regression analysis. In line with the conclusions from the preceding paper, it is suggested that formation of an abortive complex of enzyme, Mg2+, isocitrate and NADPH under catalytic cycling conditions serves to by-pass the potentially rate-limiting dissociation of NADPH from the enzyme-Mg2+-NADPH complex.
D000818:Animals; D002417:Cattle; D007521:Isocitrate Dehydrogenase / Q000378:metabolism*; D007523:Isocitrates / Q000378:metabolism; D007700:Kinetics; D008929:Mitochondria, Heart / Q000201:enzymology*; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D013050:Spectrometry, Fluorescence
D007523:Isocitrates; D009249:NADP; D007521:Isocitrate Dehydrogenase