Stepwise versus concerted oxidative decarboxylation catalyzed by malic enzyme: a reinvestigation

Abstract

The NAD-malic enzyme catalyzes the divalent metal-ion-dependent oxidative decarboxylation of L-malate to yield CO2, pyruvate, and the reduced dinucleotide. With Mg2+ as the divalent metal ion activator, primary deuterium and tritium isotope effects have been obtained with several different alternative dinucleotide substrates. The partitioning ratio of oxalacetate to malate and pyruvate has also been determined with either NAD or 3-acetylpyridine adenine dinucleotide (3-APAD). These data have been used to calculate estimates of commitment factors and intrinsic isotope effects for the NAD-malic enzyme reaction. The calculated values of the intrinsic 13C and deuterium isotope effects with NADP are similar to the previously determined values for the chicken liver malic enzyme (Grissom, C.B., & Cleland, W. W. (1988) Biochemistry 27, 2927) and suggest that the transition-state structures are similar for the Ascaris NAD- and chicken liver NADP-malic enzymes. With NAD or NADP as the dinucleotide substrate, the data are all consistent with a stepwise chemical mechanism with oxidation of L-malate at C2 preceding decarboxylation of the bound oxalacetate intermediate. However, none of the data with the alternative dinucleotide substrates, 3-acetylpyridine adenine dinucleotide and 3-pyridine aldehyde adenine dinucleotide (PAAD), can be fit with satisfaction to the various criteria that support a stepwise mechanism with NAD(P). The mechanism with 3-APAD and PAAD is likely concerted. The most likely explanation for a change in the mechanism for oxidative decarboxylation from stepwise with NAD(P) to concerted with alternative dinucleotide substrates such as 3-APAD and PAAD is a difference in the configuration of bound malate when the different dinucleotide substrates are used.