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- W2011091008 abstract "Pig heart NADP-dependent isocitrate dehydrogenase requires a divalent metal cation for catalysis. On the basis of affinity cleavage studies [Soundar and Colman (1993) J. Biol. Chem. 268, 5267] and analysis of the crystal structure of E. coli NADP−isocitrate dehydrogenase [Hurley et al. (1991) Biochemistry 30, 8671], the residues Asp253, Asp273, Asp275, and Asp279 were selected as potential ligands of the divalent metal cation in the pig heart enzyme. Using a megaprimer PCR method, the Asp at each of these positions was mutated to Asn. The wild-type and mutant enzymes were expressed in Escherichia coli and purified. D253N has a specific activity, Km values for Mn2+, isocitrate, and NADP, and also a pH−Vmax profile similar to those of the wild-type enzyme. Thus, Asp253 is not involved in enzyme function. D273N has an increased Km for Mn2+ and isocitrate with a specific activity 5% that of wild type. The D273N mutation also prevents the oxidative metal cleavage seen with Fe2+ alone in the wild-type enzyme. As compared to wild type, D275N has greatly increased Km values for Mn2+ and isocitrate, with a specific activity <0.1% that of wild type, and a large increase in pKa for the enzyme−substrate complex. D279N has only small increases in Km for Mn2+ and isocitrate, but a specific activity <0.1% that of wild type and a major change in the shape of its pH−Vmax profile. These results suggest that Asp273 and Asp275 contribute to metal binding, whereas Asp279, as well as Asp275, is critical for catalysis. Asp279 may function as the catalytic base. Using the Modeler program of Insight II, a structure for porcine NADP−isocitrate dehydrogenase was built based on the X-ray coordinates of the E. coli enzyme, allowing visualization of the metal−isocitrate site." @default.
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- W2011091008 date "2000-02-08" @default.
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- W2011091008 title "Evaluation by Site-Directed Mutagenesis of Aspartic Acid Residues in the Metal Site of Pig Heart NADP-Dependent Isocitrate Dehydrogenase" @default.
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- W2011091008 doi "https://doi.org/10.1021/bi9919753" @default.
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