title: Purification, characterization and preliminary X-ray study of fumarase from Saccharomyces cerevisiae.
abstract: 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 approximately 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 approximately 30 microM and approximately 250 microM, 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 A, space group P2(1)2(1)2(1). The unit cell contains 2 protein molecules. The crystals diffract to at least 2.6 A resolution and are suitable for X-ray structure analysis.
authors.affiliation: A.N. Bach Institute of Biochemistry, Russian Academy of Sciences, Moscow.
authors.forename: J S
authors.initials: JS
authors.lastname: Keruchenko
authors.forename: I D
authors.initials: ID
authors.lastname: Keruchenko
authors.forename: K L
authors.initials: KL
authors.lastname: Gladilin
authors.forename: V N
authors.initials: VN
authors.lastname: Zaitsev
authors.forename: N Y
authors.initials: NY
authors.lastname: Chirgadze
mesh_terms: D005591:Chemical Fractionation; D002845:Chromatography / Q000379:methods; D003460:Crystallization; D004795:Enzyme Stability; D005649:Fumarate Hydratase / Q000737:chemistry / Q000302:isolation & purification*; D007526:Isoelectric Point; D007700:Kinetics; D008970:Molecular Weight; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D014961:X-Ray Diffraction
chemical_list: D005649:Fumarate Hydratase