PMID:1917897
| Citation |
Ueda, Y, Yumoto, N, Tokushige, M, Fukui, K and Ohya-Nishiguchi, H (1991) Purification and characterization of two types of fumarase from Escherichia coli. J. Biochem. 109:728-33 |
|---|---|
| Abstract |
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. |
| Links | |
| Keywords |
Amino Acid Sequence; Escherichia coli/enzymology; Escherichia coli/genetics; Fumarate Hydratase/genetics; Fumarate Hydratase/isolation & purification; Fumarate Hydratase/metabolism; Iron/pharmacology; Kinetics; Molecular Sequence Data; Molecular Weight; Protein Conformation |
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