Evidence for an induced conformational change in the catalytic mechanism of homoisocitrate dehydrogenase for Saccharomyces cerevisiae: Characterization of the D271N mutant enzyme

Archives of Biochemistry and Biophysics
Chaonan HsuPaul F Cook

Abstract

Homoisocitrate dehydrogenase (HIcDH) catalyzes the NAD(+)-dependent oxidative decarboxylation of HIc to α-ketoadipate, the fourth step in the α-aminoadipate pathway responsible for the de novo synthesis of l-lysine in fungi. A mechanism has been proposed for the enzyme that makes use of a Lys-Tyr pair as acid-base catalysts, with Lys acting as a base to accept a proton from the α-hydroxyl of homoisocitrate, and Tyr acting as an acid to protonate the C3 of the enol of α-ketoadipate in the enolization reaction. Three conserved aspartate residues, D243, D267 and D271, coordinate Mg(2+), which is also coordinated to the α-carboxylate and α-hydroxyl of homoisocitrate. On the basis of kinetic isotope effects, it was proposed that a conformational change to close the active site and organize the active site for catalysis contributed to rate limitation of the overall reaction of the Saccharomyces cerevisiae HIcDH (Lin, Y., Volkman, J., Nicholas, K. M., Yamamoto, T., Eguchi, T., Nimmo, S. L., West, A. H., and Cook, P. F. (2008) Biochemistry47, 4169-4180.). In order to test this hypothesis, site-directed mutagenesis was used to change D271, a metal ion ligand and binding determinant for MgHIc, to N. The mutant enzyme was characterized us...Continue Reading

References

Sep 1, 2006·Cell Biochemistry and Biophysics·Hengyu XuPaul F Cook
Sep 12, 2007·Bioinformatics·M A LarkinD G Higgins
Dec 19, 2007·Bioorganic & Medicinal Chemistry·Takashi Yamamoto, Tadashi Eguchi
Jun 3, 2010·Acta Crystallographica. Section D, Biological Crystallography·Radhika Malik, Ronald E Viola

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