Critical roles of conserved carboxylic acid residues in pigeon cytosolic NADP+-dependent malic enzyme.
Malic enzyme catalyses the reduction of NADP+ to NADPH and the decarboxylation of L-malate to pyruvate through a general acid/base mechanism. Previous kinetic and structural studies differ in their interpretation of the amino acids responsible for the general acid/base mechanism. To resolve this discrepancy, we used site-directed mutagenesis and kinetic analysis to study four conserved carboxylic amino acids. With the D257A mutant, the Km for Mn2+ and the kcat decreased relative to those of the wild-type by sevenfold and 28-fold, respectively. With the E234A mutant, the Km for Mg2+ and L-malate increased relative to those of the wild-type by 87-fold and 49-fold, respectively, and the kcat remained unaltered, which suggests that the E234 residue plays a critical role in bivalent metal ion binding. The kcat for the D235A and D258A mutants decreased relative to that of the wild-type by 7800-fold and 5200-fold, respectively, for the overall reaction, by 800-fold and 570-fold, respectively, for the pyruvate reduction partial reaction, and by 371-fold and 151-fold, respectively, for the oxaloacetate decarboxylation. The activities of the overall reaction and the pyruvate reduction partial reaction of the D258A mutant were rescued by the presence of 50 mM sodium azide. In contrast, small free acids did not have a rescue effect on the activities of the E234A, D235A, and D257A mutants. These data suggest that D258 may act as a general base to extract the hydrogen of the C2 hydroxy group of L-malate with the aid of D235-chelated Mn2+ to polarize the hydroxyl group.
Chang
Shuo-Chin
SC
Department of Biochemistry, National Defense Medical Center, Taipei, Taiwan.
Lin
Kuan-Yu
KY
Chen
Yu-Jung
YJ
Lai
Chin-Hung
CH
Chang
Gu-Gang
GG
Chou
Wei-Yuan
WY
D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D002264:Carboxylic Acids / Q000737:chemistry / Q000378:metabolism*; D010856:Columbidae; D017124:Conserved Sequence; D003600:Cytosol / Q000201:enzymology*; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D016297:Mutagenesis, Site-Directed
D002264:Carboxylic Acids; D008291:Malate Dehydrogenase; C031917:malate dehydrogenase (oxaloacetate-decarboxylating) (NADP+)