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Thr373, Asp375, and Lys260 are in the coenzyme site of porcine NADP-dependent isocitrate dehydrogenase.
Arch Biochem Biophys. 2006 Jun 15; 450(2):183-90.AB

Abstract

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.

Authors+Show Affiliations

Department of Chemistry and Biochemistry, University of Delaware, Newark, DE 19716, USA.No affiliation info available

Pub Type(s)

Journal Article
Research Support, N.I.H., Extramural

Language

eng

PubMed ID

16712772

Citation

Lee, Peychii, and Roberta F. Colman. "Thr373, Asp375, and Lys260 Are in the Coenzyme Site of Porcine NADP-dependent Isocitrate Dehydrogenase." Archives of Biochemistry and Biophysics, vol. 450, no. 2, 2006, pp. 183-90.
Lee P, Colman RF. Thr373, Asp375, and Lys260 are in the coenzyme site of porcine NADP-dependent isocitrate dehydrogenase. Arch Biochem Biophys. 2006;450(2):183-90.
Lee, P., & Colman, R. F. (2006). Thr373, Asp375, and Lys260 are in the coenzyme site of porcine NADP-dependent isocitrate dehydrogenase. Archives of Biochemistry and Biophysics, 450(2), 183-90.
Lee P, Colman RF. Thr373, Asp375, and Lys260 Are in the Coenzyme Site of Porcine NADP-dependent Isocitrate Dehydrogenase. Arch Biochem Biophys. 2006 Jun 15;450(2):183-90. PubMed PMID: 16712772.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Thr373, Asp375, and Lys260 are in the coenzyme site of porcine NADP-dependent isocitrate dehydrogenase. AU - Lee,Peychii, AU - Colman,Roberta F, Y1 - 2006/05/02/ PY - 2005/12/08/received PY - 2006/03/31/revised PY - 2006/04/02/accepted PY - 2006/5/23/pubmed PY - 2006/8/19/medline PY - 2006/5/23/entrez SP - 183 EP - 90 JF - Archives of biochemistry and biophysics JO - Arch. Biochem. Biophys. VL - 450 IS - 2 N2 - 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. SN - 0003-9861 UR - https://www.unboundmedicine.com/medline/citation/16712772/Thr373_Asp375_and_Lys260_are_in_the_coenzyme_site_of_porcine_NADP_dependent_isocitrate_dehydrogenase_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0003-9861(06)00139-1 DB - PRIME DP - Unbound Medicine ER -