Author: Ueda, Y.; Yumoto, N.; Tokushige, M.; Fukui, K.; Ohya-Nishiguchi, H.
Description: Two distinct types of fumarase were purified to homogeneity from aerobically grown Escherichia coli W cells. The amino acid sequences of their NH2-terminals suggest that the two enzymes are the products of the fumA gene (FUMA) and fumC gene (FUMC), respectively. FUMA was separated from FUMC by chromatography on a Q-Sepharose column, and was further purified to homogeneity on Alkyl-Superose, Mono Q, and Superose 12 columns. FUMA is a dimer composed of identical subunits (Mr = 60,000). Although the activity of FUMA rapidly decreased during storage, reactivation was attained by anaerobic incubation with Fe2+ and thiols. Studies on the inactivation and reactivation of FUMA suggested that oxidation and the concomitant release of iron inactivated the enzyme in a reversible manner. While the inactivated FUMA was EPR-detectable, through a signal with g perpendicular = 2.02 and g = 2.00, the active enzyme was EPR-silent. These results suggested FUMA is a member of the 4Fe-4S hydratases represented by aconitase. After the separation of FUMC from FUMA, purification of the former enzyme was accomplished by chromatography on Phenyl-Superose and Matrex Gel Red A columns. FUMC was stable, Fe-independent and quite similar to mammalian fumarases in enzymatic properties.
Subject headings: Amino Acid Sequence; Escherichia coli; Fumarate Hydratase; Iron; Kinetics; Molecular Sequence Data; Molecular Weight; Protein Conformation; E coli
Publication year: 1991
Journal or book title: Journal of Biochemistry
Volume: 109
Issue: 5
Pages: 728-733
Find the full text: https://www.jstage.jst.go.jp/article/biochemistry1922/109/5/109_5_728/_pdf
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Serial number: 3865
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