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 Km values for Mn2+ and l-malate similar to those of wild-type. The Km value for NADP+ of K162A is identical to that of wild-type. However, K162A demonstrated a 235-fold decrease in the kcat 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 kcat value. However, its Km 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+.