Purification and Properties of Fumarate Reductase from Baker's Yeast

Abstract

Fumarate reductase was highly purified from the cytosol fraction of the cells of baker's yeast, which were anaerobically cultured, with a yield of 6% by 7 steps. The purified enzyme is homogeneous in disc-gel electrophoresis, and has a molecular weight of 58,800. Its isoelectric point is 5.3. The enzyme contains 1 molecule of non-covalently bound FAD per molecule of protein. Non-heme iron was not detected. The amino acid composition was determined. The spectra of the enzyme showed absorption peaks at 273, 380 and 456 nm with shoulders at 370, 440 and 478 nm. The fluorescence spectra showed excitation peaks at 452 and 368 nm and an emission peak at 500 nm. Double-reciprocal plots of the reaction rate against the FADH2 and FMNH2 concentrations curved upwards. The hill coefficient values and S0.5for FADH2 and FMNH2 were estimated to be 1.6 and 0.21 μm, and 1.5 and 0.38 μm, respectively. The Km for fumarate was 0.2 mM. © 1982, Japan Society for Bioscience, Biotechnology, and Agrochemistry. All rights reserved.