Expression and mutagenesis of mammalian cytosolic NADP+-specific isocitrate dehydrogenase.
Rat liver cytosolic NADP+-specific isocitrate dehydrogenase (IDP2) was expressed in bacteria as a fusion protein with maltose binding protein (MBP). High levels of expression were obtained. The fusion protein was purified from bacterial lysates by affinity chromatography with an amylose resin and found to be catalytically active. IDP2 was separated from MBP by cleavage with protease Xa and purified to homogeneity by FPLC anion-exchange chromatography. A specific activity of 56.3 units/mg and respective apparent Km values for dl-isocitrate and NADP+ of 9.7 +/- 2.9 microM and 11.5 +/- 0.2 microM were obtained for the purified enzyme. These values are similar to those previously reported for cytosolic isocitrate dehydrogenase isolated from a variety of tissues. Evolutionarily conserved arginine residues implicated in substrate binding were changed to glutamate residues using PCR based site-directed mutagenesis of the bacterial fusion plasmid. Mutant enzymes containing residue changes of R100E, R109E, R119E, or R132E were expressed, purified, and characterized by initial rate kinetic analyses. The R119E and R109E mutant enzymes exhibited respective 15- and 31-fold increases in Km values for dl-isocitrate relative to the wild-type enzyme. In contrast, Km values for NADP+ were, respectively, unchanged and increased 9-fold. The most significant reductions in kcat/Km values were obtained for the R100E, R109E, and R132E enzymes. These results suggest that substrate binding residues are highly conserved between bacterial and mammalian enzymes despite low overall homology.
Jennings
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GT
Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284-7760, USA.
Minard
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KI
McAlister-Henn
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L
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D003600:Cytosol / Q000201:enzymology*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D007521:Isocitrate Dehydrogenase / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000378:metabolism; D010957:Plasmids; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D009249:NADP; D007521:Isocitrate Dehydrogenase; C039355:isocitrate dehydrogenase (NADP+)