Lysine 199 is the general acid in the NAD-malic enzyme reaction

Site-directed mutagenesis was used to change K199 in the Ascaris suum NAD-m alic enzyme to A and R and Y126 to F. The K199A mutant enzyme gives a 10(5) -fold decrease in V and a 10(6)-fold decrease in V/K-malate compared to the WT enzyme. In addition, the ratio for partitioning of the oxalacetate inte rmediate toward pyruvate and malate changes from a value of 0.4 for the WT enzyme to 1.6 for K199A, and repeating the experiment with A-side NADD give s isotope effects of 3 and 1 for the WT and K199A mutant enzymes, respectiv ely. The K199R mutant enzyme gives only a factor of 10 decrease in V, and t he pK for the general acid in this mutant enzyme has increased from 9 for t he WT enzyme to > 10 for the K199R mutant enzyme. Tritium exchange from sol vent into pyruvate is catalyzed by the WT enzyme, but not by the K199A muta nt enzyme. The Y126F mutant enzyme gives a 10(3)-fold decrease in V. The ox alacetate partition ratio and isotope effect on oxalacetate reduction for t he Y126F mutant enzyme are identical, within error, to those measured for t he WT enzyme. Thus, Y126 is important to the overall reaction, but its role at present is unclear. Data are consistent with K199 functioning as the ge neral acid that protonates C3 of enolpyruvate to generate the pyruvate prod uct in the malic enzyme reaction.