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

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 α-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 μM; Km (α-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 (α/β) 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.