Isotope effect studies of isocitrate dehydrogenase

The carbon isotope effect (k13/k12) on the oxidative decarboxylation of isocitrate catalyzed by isocitrate dehydrogenase with Mg2+ as a cofactor is 0.9989 indicating that decarboxylation is not the rate limiting step of the reaction. When Ni2+ is substituted for Mg2+, the enzyme catalyzed reaction exhibits a slightly higher carbon isotope effect of 1.0050. This increase due to the change in metal ion is interpreted as a decrease in the rate of decarboxylation relative to that of the rate determining step. The hydrogen isotope effect (k/sup H//k/sup D/) on V/K with Mg2+ as a cofactor is 0.76, which was somewhat surprising but not unique. Inverse isotope effects have been reported for the binding of substrates and in organic reactions where steric requirements are very stringent. Therefore an inverse isotope effect for this reaction is an expression of the isotope effect on the binding of substrate. The hydrogen isotope effect on V/sub max/ with either Mg2+ or Ni2+ as a cofactor is very small. However, at pH 5.5 the isotope effect is 1.51. An isotope effect on the equilibrium constant (K/sub eq//sup H//K/sub eq//sup D/) of 1.11 was determined. This is in agreement with theoretical calculations and results from the fact that there is a thermodynamic preference for the deuterium being in the molecule with the highest zero point energy, isocitrate in this case. The results indicate that the release of isocitrate from the enzyme-substrate complex is slower than catalysis but not as slow as TPN release. The role of the metal is to stabilize the intermediates as well as destabilize the transition states