Purification, characterization and preliminary X-ray study of fumarase from Saccharomyces cerevisiae
| Autor: | Gladilin Kl, Nickolai Y. Chirgadze, Vyacheslav N. Zaitsev, Irina D. Keruchenko, Jan S. Keruchenko |
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| Rok vydání: | 1992 |
| Předmět: |
Saccharomyces cerevisiae
Biophysics Fractionation Chemical Fractionation Biochemistry Fumarate Hydratase X-Ray Diffraction Affinity chromatography Structural Biology Enzyme Stability Isoelectric Point Molecular Biology Equilibrium constant chemistry.chemical_classification Chromatography Molecular mass biology biology.organism_classification Molecular Weight Kinetics Enzyme Isoelectric point chemistry Fumarase Crystallization |
| Zdroj: | Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology. 1122:85-92 |
| ISSN: | 0167-4838 |
| DOI: | 10.1016/0167-4838(92)90131-v |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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