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J. Biol. Chem., Vol. 280, Issue 6, 4469-4475, February 11, 2005

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Kinetic Properties and Metabolic Contributions of Yeast Mitochondrial and Cytosolic NADP⁺-specific Isocitrate Dehydrogenases^*

Veronica Contreras-Shannon, An-Ping Lin, Mark T. McCammon, and Lee McAlister-Henn

From the Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78229-3900

To compare kinetic properties of homologous isozymes of NADP⁺-specific isocitrate dehydrogenase, histidine-tagged forms of yeast mitochondrial (IDP1) and cytosolic (IDP2) enzymes were expressed and purified. The isozymes were found to share similar apparent affinities for cofactors. However, with respect to isocitrate, IDP1 had an apparent K_m value 7-fold lower than that of IDP2, whereas, with respect to -ketoglutarate, IDP2 had an apparent K_m value 10-fold lower than that of IDP1. Similar K_m values for substrates and cofactors in decarboxylation and carboxylation reactions were obtained for IDP2, suggesting a capacity for bidirectional catalysis in vivo. Concentrations of isocitrate and -ketoglutarate measured in extracts from the parental strain were found to be similar with growth on different carbon sources. For mutant strains lacking IDP1, IDP2, and/or the mitochondrial NAD⁺-specific isocitrate dehydrogenase (IDH), metabolite measurements indicated that major cellular flux is through the IDH-catalyzed reaction in glucose-grown cells and through the IDP2-catalyzed reaction in cells grown with a nonfermentable carbon source (glycerol and lactate). A substantial cellular pool of -ketoglutarate is attributed to IDH function during glucose growth, and to both IDP1 and IDH function during growth on glycerol/lactate. Complementation experiments using a strain lacking IDH demonstrated that overexpression of IDP1 partially compensated for the glutamate auxotrophy associated with loss of IDH. Collectively, these results suggest an ancillary role for IDP1 in cellular glutamate synthesis and a role for IDP2 in equilibrating and maintaining cellular levels of isocitrate and -ketoglutarate.

Received for publication, September 3, 2004 , and in revised form, December 1, 2004.

^* This work was supported by National Institutes of Health Grants AG17477 and GM51265. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Dept. of Microbiology and Immunology, South Texas Centers for Biology in Medicine, University of Texas Health Science Center, San Antonio, TX 78245.

To whom correspondence should be addressed. Tel.: 210-567-3782; Fax: 210-567-6595; E-mail: henn{at}uthscsa.edu.

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