PURIFICATION, CHARACTERIZATION AND PRELIMINARY-X-RAY STUDY OF FUMARASE FROM SACCHAROMYCES-CEREVISIAE

Fumarase (fumarate hydratase, EC 4.2.1.2) from Saccharomyces cerevisiae has been purified to homogeneity by a method including acetone fractionation, DEAE ion-exchange and dye-sorbent affinity chromatography. The suggested method allows fumarase purification with a yield higher than 60% and may be used to obtain large enzyme quantities. The native protein consists of four subunits with a almost-equal-to 50 kDa molecular mass each and has an isoelectric point at pH 6.5 +/- 0.3. The equilibrium constant for fumarate hydration is about 4.3 (25-degrees-C, pH 7.5), the Michaelis constants for fumarate and 1-malate are almost-equal-to 30-mu-M and almost-equal-to 250-mu-M, respectively. The enzyme is activated by substrates and multivalent anions, the activation seems to be of a non-competitive type. The fumarase complex with meso-tartaric acid has been crystallized by the vapor diffusion method. The unit cell parameters are a = 93.30, b = 94.05 and c = 106.07 angstrom, space group P2(1)2(1)2(1). The unit cell contains 2 protein molecules. The crystals diffract to at least 2.6 angstrom resolution and are suitable for X-ray structure analysis.