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

Alanine-scanning site-directed mutagenesis was carried out on all conserved lysine residues of pigeon cytosolic NADP(+)-dependent malic enzyme. Only t wo mutant enzymes, K162A and K340A, showed significant effect on their kine tic parameters, Both mutant enzymes have K-m values for Mn2+ and L-malate s imilar to those of wild-type. The K-m value for NADP(+) of K162A is identic al 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 suggest ed that the side chain of K162 is important for the enzyme catalytic reacti on. We propose that the epsilon-amino group of K162 may serve as a general acid to protonate the 3-carbon of enolpyruvate after decarboxylation. The K 340A mutant demonstrated no effect on the k(cat) value, However, its K-m va lue for NADP(+) was increased by a factor of 65 (225.7 +/- 5.07 vs 3.49 +/- 0.05 mu M). We propose that the NADP(+) specificity is determined by the e lectrostatic interaction between the epsilon-amino group of K340 and 2'-pho sphate of NADP(+). (C) 2000 Academic Press.