Determination of the mechanism of human malic enzyme with natural and alternate dinucleotides by isotope effects

Human malic enzyme was studied by steady state kinetics, deuterium isotope effects, and C-13 isotope effects with both the physiological dinucleotide cofactor and several alternate cofactors. The log V vs pH profile with NAD revealed two pK(a) values too close to be separately determined, but with a n average value of 7.33. The log V/K vs pH profile with NAD revealed two pK (a) values at 7.4 and 5.6. Deuterium and C-13 isotope effects indicate that the mechanism of human malic enzyme is stepwise with both NAD and epsilon NAD, but that hyperconjugation in the transition state for hydride transfer is detectable only with the former. With thioNAD and A-PAD, the isotope ef fects do not clearly indicate whether the mechanism is stepwise or concerte d. The intrinsic C-13 isotope effect for decarboxylation was calculated to be 1.0485 by measurement of the partition ratio of oxaloacetate in the pres ence of NADH and human malic enzyme (decarboxylation to pyruvate/ reduction to malate = 2.33). The isotope effect and partitioning data suggest that t he energy barrier for decarboxylation of oxaloacetate is not as high relati ve to the barrier for reduction of oxaloacetate as with the chicken liver e nzyme. (C) 2001 Elsevier Science.