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 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.
Andi
Babak
B
Department of Chemistry and Biochemistry, University of Oklahoma, 620 Parrington Oval, Norman, Oklahoma 73019, USA.
West
Ann H
AH
Cook
Paul F
PF
D015086:2,6-Dichloroindophenol / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D003065:Coenzyme A / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D007700:Kinetics; D018832:Molecular Chaperones / Q000378:metabolism; D007652:Oxo-Acid-Lyases / Q000032:analysis / Q000378:metabolism*; D020673:Protein Renaturation; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
D018832:Molecular Chaperones; D015086:2,6-Dichloroindophenol; C021365:homocitrate synthase; D007652:Oxo-Acid-Lyases; D003065:Coenzyme A