Use of isotope effects to determine the chemical mechanism of oxidative decarboxylases: NADP malic enzyme and NADP isocitrate dehydrogenase

The chemical mechanism of the NADP-linked oxidative decarboxylases chicken liver malic enzyme and pig heart isocitrate dehydrogenase has been examined using carbon and deuterium isotope effects and their variation with pH, metal ions, and solution viscosity. The following 13C isotope effects on V/K for malate are observed with the stated metal ion at pH 8.0 and 250C: Mg2+, 1.0336; Mn2+, 1.0365; Cd2+, 1.0366; Zn2+, 1.03373; Co2+, 1.0283; and Ni2+, 1.025. The 13C isotope effect on nonenzymatic decarboxylation of dibasic oxalacetate at 250C and the stated metal ion is : Mg2+, 1.0489; Mn2+, 1.0505; Ni2+, 1.044; Cd2+, 1.0492; Zn2+, 1.0504; and Co2+, 1.0480. By quantitating the partitioning of the 2-ketocarboxylic acid reaction intermediate between reverse hydride transfer and decarboxylation, it is possible to solve for the intrinsic isotope effects in both reactions. With malic enzyme activated by Mg2+, the partitioning ratio of oxalacetate between pyruvate and malate formation is 0.47. This gives an intrinsic deuterium and 13C isotope effect of 5.6 and 1.0493, respectively. The 13C isotope effect for the Mg2+ catalyzed nonenzymatic decarboxylation of oxalacetate is 1.0489, thereby suggesting a similarity of transition states between the enzymatic and nonenzymatic processes. The observed 13C primary isotope effect for isocitrate in the isocitrate dehydrogenase reaction is obscured by the stickiness of isocitrate at neutral pH. At low pH, the external commitment is eliminated and the observed 13C isotope effect increases to a limiting value of 1.0353. The pK of the pH dependence of 13(V/K) is 4.7