Lysine residues 162 and 340 are involved in the catalysis and coenzyme binding of NADP(+)-dependent malic enzyme from pigeon.

Abstract Alanine-scanning site-directed mutagenesis was carried out on all conserved lysine residues of pigeon cytosolic NADP + -dependent malic enzyme. Only two mutant enzymes, K162A and K340A, showed significant effect on their kinetic parameters. Both mutant enzymes have K m values for Mn 2+ and l -malate similar to those of wild-type. The K m value for NADP + of K162A is identical to that of wild-type. However, K162A demonstrated a 235-fold decrease in the k cat value (0.17 ± 0.01 vs 40.0 ± 1.3 s −1 ). These data suggested that the side chain of K162 is important for the enzyme catalytic reaction. We propose that the ϵ-amino group of K162 may serve as a general acid to protonate the 3-carbon of enolpyruvate after decarboxylation. The K340A mutant demonstrated no effect on the k cat value. However, its K m value for NADP + was increased by a factor of 65 (225.7 ± 5.07 vs 3.49 ± 0.05 μM). We propose that the NADP + specificity is determined by the electrostatic interaction between the ϵ-amino group of K340 and 2′-phosphate of NADP + .