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 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 epsilon-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 microM). We propose that the NADP(+) specificity is determined by the electrostatic interaction between the epsilon-amino group of K340 and 2'-phosphate of NADP(+).
Kuo
C C
CC
Graduate Institutes of Life Sciences and Biochemistry, National Defense Medical Center, Taipei, Taiwan, Republic of China.
Tsai
L C
LC
Chin
T Y
TY
Chang
G G
GG
Chou
W Y
WY
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D003001:Cloning, Molecular; D010856:Columbidae; D017124:Conserved Sequence; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008239:Lysine; D008291:Malate Dehydrogenase / Q000737:chemistry* / Q000378:metabolism*; D008293:Malates / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D008293:Malates; D011994:Recombinant Proteins; D008345:Manganese; D009249:NADP; D008291:Malate Dehydrogenase; C031917:malate dehydrogenase (oxaloacetate-decarboxylating) (NADP+); D008239:Lysine