Mapping the active site topography of the NAD-malic enzyme via alanine-scanning site-directed mutagenesis.
The NAD-malic enzyme cDNA has been subcloned into the pQE expression vector, expressed with a six-His tag, and purified. The His-tagged enzyme is purified by a combination of Ni-NTA and orange A agarose column chromatography with a yield of 45% and an estimated purity of >90%. The tag and linker have no effect on the kinetic parameters of the enzyme compared to the wild-type enzyme. Alanine-scanning site-directed mutagenesis has been carried out on all of the conserved neutral acid residues of the NAD-malic enzyme from Ascaris suum. Data obtained confirm the predicted role of D178 and D295 in metal ion binding, the likely role of D294, D361, and E440 in the NAD binding site, and the role of E58 and D272 in malate binding. Decreases in V/E(t) by 10(4)-fold and in V/K(malate)E(t) by 10(7)-fold, when D295 is changed to alanine, suggest that it is a likely candidate for the general base that accepts a proton from the malate hydroxyl in the oxidation step.
Karsten
W E
WE
Department of Chemistry and Biochemistry, University of Oklahoma, Norman 73019, USA.
Chooback
L
L
Liu
D
D
Hwang
C C
CC
Lynch
C
C
Cook
P F
PF
D000409:Alanine / Q000235:genetics*; D000818:Animals; D017165:Ascaris suum / Q000201:enzymology; D001665:Binding Sites / Q000235:genetics; D002413:Cations, Divalent; D017124:Conserved Sequence; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008293:Malates / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D002413:Cations, Divalent; D008293:Malates; D011994:Recombinant Proteins; C030298:malic acid; D008291:Malate Dehydrogenase; C046446:malate dehydrogenase-(oxaloacetate-decarboxylating) (NAD+); D000409:Alanine