Proper positioning of the nicotinamide ring is crucial for the Ascaris suum malic enzyme reaction

The mitochondrial NAD-malic enzyme catalyzes the oxidative decarboxylation of malate to pyruvate and CO2. The role of the dinucleotide substrate in oxidative decarboxylation is probed in this study using site-directed mutagenesis to change key residues that line the dinucleotide binding site. Mutant enzymes were characterized using initial rate kinetics, and isotope effects were used to obtain information on the contribution of these residues to binding energy and catalysis. Results obtained for the N479 mutant enzymes indicate that the hydrogen bond donated by N479 to the carboxamide side chain of the nicotinamide ring is important for proper orientation in the hydride transfer step. The stepwise oxidative decarboxylation mechanism observed for the wt enzyme changed to a concerted one, which is totally rate limiting, for the N479Q mutant enzyme. In this case, it is likely that the longer glutamine side chain causes reorientation of malate such that it binds in a conformation that is optimal for concerted oxidative decarboxylation. Converting N479 to the shorter serine side chain gives very similar values of KNAD, Kmalate, and isotope effects relative to wt, but V/Et is decreased 2 000-fold. Data suggest an increased freedom of rotation, resulting in nonproductively bound cofactor. Changes were also made to two residues, S433 and N434, which interact with the nicotinamide ribose of NAD. In addition, N434 donates a hydrogen bond to the beta-carboxylate of malate. The KNAD for the S433A mutant enzyme increased by 80-fold, indicating that this residue provides significant binding affinity for the dinucleotide. With N434A, the interaction of the residue with malate is lost, causing the malate to reorient itself, leading to a slower decarboxylation step. The longer glutamine and methionine side chains stick into the active site and cause a change in the position of malate and/or NAD resulting in more than a 104-fold decrease in V/Et for these mutant enzymes. Overall, data indicate that subtle changes in the orientation of the cofactor and substrate dramatically influence the reaction rate.