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Arch Biochem Biophys. 2004 Jan 15;421(2):243-54.

Stabilization and characterization of histidine-tagged homocitrate synthase from Saccharomyces cerevisiae.

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Department of Chemistry and Biochemistry, University of Oklahoma, 620 Parrington Oval, Norman, Oklahoma 73019, USA.


Histidine-tagged homocitrate synthase from Saccharomyces cerevisiae was purified to about 98% using a Ni-NTA resin and stabilized using a combination of 100 mM guanidine hydrochloride, 100 mM alpha-cyclodextrin, and 600 mM ammonium sulfate. The enzyme was assayed using dichlorophenol indophenol (DCPIP) as an oxidant to oxidize the CoASH produced in the reaction. A stoichiometry of 1:1 was obtained between DCPIP and CoASH. Kinetic parameters for the stable enzyme at pH 7.5 are: Km (AcCoA), 24 microM: Km (alpha-kg), 1.3 mM; and kcat, 37 min(-1). The enzyme, in the absence of reactants, self-associates, as suggested by size exclusion chromatography. Fluorescence and circular dichroic spectra suggested a partially exposed tryptophan residue and a mixed (alpha/beta) secondary structure for the enzyme. Fluorescence quenching studies with KI, CsCl, and acrylamide suggest that the microenvironment around the single tryptophan residue of the enzyme has some positive charge.

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