METAL-ION ACTIVATOR EFFECTS ON INTRINSIC ISOTOPE EFFECTS FOR HYDRIDE TRANSFER AND DECARBOXYLATION IN THE REACTION CATALYZED BY THE NAD-MALIC ENZYME FROM ASCARIS-SUUM

The mechanism of the oxidative decarboxylation reaction catalyzed by the NAD-malic enzyme from Ascaris suum has been examined with several different divalent metal ion activators and dinucleotide substrates. Primary deuterium and tritium isotope effects have been obtained and, in combination with the partitioning ratios of the oxalacetate intermediate to malate and pyruvate, have been used to calculate commitment factors, intrinsic deuterium isotope effects on the hydride transfer step, and intrinsic C-13 isotope effects for the decarboxylation step. A survey of malate analogs has been undertaken to define the geometry of the active site and to identify functional groups on malate important for substrate binding. With NAD as dinucleotide substrate, a direct correlation between the size of the divalent metal ion activator and the intrinsic deuterium isotope effect is observed. An isotope effect significantly greater than the semiclassical limit is seen when Cd2+ is the metal ion activator, indicating a substantial tunneling contribution. The primary intrinsic C-13 isotope effect on the decarboxylation step increases over the series Mg2+ < Mn2+ < Cd2+, which is in contrast to the equal isotope effects measured for these metal ions for the nonenzymatic decarboxylation of oxalacetate [Grissom, C. B., and Cleland, W. W. (1986) J. Am. Chem. Sec. 108, 5582]. With Mn2+ or Cd2+ as the divalent metal ion activator, the data support a stepwise mechanism for the enzymatic oxidative decarboxylation with NAD as the dinucleotide substrate, but a change to a concerted mechanism is indicated with more redox-positive dinucleotide substrates as suggested previously with Mg2+ as activator [Karsten, W. E., and Cook, P. F. (1994) Biochemistry 33, 2096]. A survey of malate inhibitory analogs suggests the enzyme is quite forgiving with respect to functional group substitutions at the beta-carbon and also indicates that the metal ion provides a major determinant for substrate binding.