Purification and characterization of two types of fumarase from Escherichia coli.
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.
Ueda
Y
Y
Department of Chemistry, Faculty of Science, Kyoto University.
Yumoto
N
N
Tokushige
M
M
Fukui
K
K
Ohya-Nishiguchi
H
H
D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005649:Fumarate Hydratase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D007501:Iron / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011487:Protein Conformation
D007501:Iron; D005649:Fumarate Hydratase