Thr373, Asp375, and Lys260 are in the coenzyme site of porcine NADP-dependent isocitrate dehydrogenase.
Thr(373), Lys(374), Asp(375), and Lys(260) were chosen as site-directed mutagenesis targets within porcine NADP-dependent isocitrate dehydrogenase based on structurally corrected sequence alignment among prokaryotic and eukaryotic NADP-isocitrate dehydrogenases. Wild-type and all mutant enzymes were expressed in Escherichia coli and purified to homogeneity. These mutations do not alter the secondary structure or dimerization state of the mutants. The D375N and K260Q mutants exhibit, respectively, a 15- and 28-fold increase in K(m) for NADP, along with marked decreases in V(max) as compared to wild-type enzyme. In contrast, replacing Lys(374), which was previously proposed to contribute to apparent coenzyme affinity, does not change the enzyme's kinetic parameters. T373S exhibits similar kinetic parameters to those of wild-type while T373A and T373V mutations reduce the V(max) values of the resulting enzymes to 1 and 20%, respectively of that of wild-type. We conclude that a hydroxyl group at position 373 is required for effective enzyme function and that Asp(375) and Lys(260) are critical amino acids contributing to coenzyme affinity as well as catalysis by porcine NADP-isocitrate dehydrogenase.
Lee
Peychii
P
Department of Chemistry and Biochemistry, University of Delaware, Newark, DE 19716, USA.
Colman
Roberta F
RF
D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid / Q000235:genetics; D001665:Binding Sites; D007521:Isocitrate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008239:Lysine / Q000235:genetics; D008929:Mitochondria, Heart / Q000201:enzymology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013552:Swine; D013912:Threonine / Q000235:genetics
D011994:Recombinant Proteins; D013912:Threonine; D001224:Aspartic Acid; D009249:NADP; D007521:Isocitrate Dehydrogenase; D008239:Lysine