Kinetic Mechanism of Escherichia coli Isocitrate Dehydrogenase

The kinetic mechanism of the NADP-dependent isocitrate dehydrogenase of Escherichia coli was investigated using initial steady-state kinetic analyses. Kinetic coefficients, obtained using natural and alternative substrates with the wild-type and two mutant enzymes (S113L and S113N), suggest that the forward reaction [the oxidative decarboxylation of (2R, 3S)-isocitrate by NADP] of the wild-type enzyme is a steady-state random mechanism, with catalysis more rapid than product release. The mechanism of the wild-type enzyme becomes rapid-equilibrium random when an alternative substrate [(2R)-malate or NAD] is used. The mutant enzymes always display rapid-equilibrium random kinetics, and for each enzyme the apparent dissociation constant of each substrate from the binary complex [K_ia = E·A/(EA)] is similar to its apparent dissociation constant from the Michaelis complex [K_a = (EB)·A/(EAB)], which suggests that the binding of one substrate is independent of the binding of the second. When the wild-type enzyme catalyzes the forward reaction, the apparent dissociation constant, K_iISo, is equal to its equilibrium dissociation constant, K_dIso, determined from equilibrium binding studies. However, the apparent dissociation constant of the cofactor, K_iNADP, is far smaller than its equilibrium dissociation constant, K_dNADP. This is consistent with the proposed mechanism, because simulations show that when catalysis is steady-state and product release is rate-limiting, K_iNADP and K_NADP will be far smaller than K_dNADP, while K_iIso and K_Iso remain similar to K_dIso. Product inhibition studies support the steady-state random mechanism of the wild-type enzyme. The rapid-equilibrium random mechanisms of the mutant enzymes provide evidence for the existence of E·Iso·NADPH and E·αKg·NADP abortive complexes and demonstrate that α-ketoglutarate and NADPH each bind to the free enzyme. Initial steady-state rate and product inhibition studies of the reverse reaction indicate a random addition of α-ketoglutarate and NADPH, with CO₂ possibly binding last. Dead-end inhibition studies, using tricarballylate (propane-1,2,3-tricarboxylic acid) as an analog of isocitrate and adenosine 2′,5′-diphosphate as an analog of NADP, are incompatible with ordered mechanisms in either direction.