Escherichia coli fumarase A catalyzes the isomerization of enol and keto oxalacetic acid

Abstract

Fumarase A, a product of the fumA gene of Escherichia coli, has been found to catalyze the isomerization of enol to keto oxalacetic acid (OAA) in addition to catalyzing the fumarase reaction. The kcat/Km for the isomerization is almost identical to that for the fumarase reaction. The isomerization reaction apparently takes place at the same active site as the fumarase reaction since both reactions show a similar sensitivity to inactivation by O2, both reactions are strongly inhibited by 2-hydroxy-3-nitropropionate, and the isomerization reaction is inhibited by fumarate and malate. The isomerization requires the presence of a [4Fe-4S] or [3Fe-4S] cluster, perhaps for structural rather than catalytic reasons. Hydration of enol OAA to the gem diol has been ruled out as a possible mechanism of isomerization on the basis of the preservation of the oxygen on carbon 2 and the position of protonation on carbon 3. The isomerization is not stereospecific in the position of protonation at carbon 3 but appears to be stereoselective, with protonation preferentially occurring in the 3-pro-S position. Porcine fumarase, isopropyl malate isomerase, and dihydroxyacid dehydratase do not catalyze this isomerization. Fumarase A and aconitase, two enzymes with 4Fe-4S clusters that bind a linear 4-carbon dicarboxylic acid moiety in the trans conformation during their normal hydro-lyase reaction, do catalyze this isomerization.

Enzyme promiscuity, metabolite damage, and metabolite damage control systems of the tricarboxylic acid cycle

“…Proteins with OAA enol‐keto tautomerase (OAT, EC 5.3.2.2) activity from several prokaryotes and eukaryotes have been reported [58,61,62]. It was demonstrated that OAT enzymes, in the presence of a (keto) OAA‐metabolizing enzyme, protect SDH from enol‐OAA inactivation that would otherwise occur as a result of promiscuous malate oxidation [56].…”

“…It was demonstrated that OAT enzymes, in the presence of a (keto) OAA‐metabolizing enzyme, protect SDH from enol‐OAA inactivation that would otherwise occur as a result of promiscuous malate oxidation [56]. Interestingly, in mammals, the TCA cycle hydro‐lyase enzyme aconitase has OAT activity [63], while in E. coli , another TCA cycle hydro‐lyase enzyme fumarate hydratase (FH) has OAT activity [62]. For both enzymes, OAT activity is catalyzed with nearly the same catalytic efficiency as their respective hydro‐lyase activities [62].…”

“…Interestingly, in mammals, the TCA cycle hydro‐lyase enzyme aconitase has OAT activity [63], while in E. coli , another TCA cycle hydro‐lyase enzyme fumarate hydratase (FH) has OAT activity [62]. For both enzymes, OAT activity is catalyzed with nearly the same catalytic efficiency as their respective hydro‐lyase activities [62]. OAT activity may be related to an 4Fe‐4S cluster that is present in E. coli FH and mammalian aconitase, but not E. coli aconitase and mammalian FH [62].…”

“…For both enzymes, OAT activity is catalyzed with nearly the same catalytic efficiency as their respective hydro‐lyase activities [62]. OAT activity may be related to an 4Fe‐4S cluster that is present in E. coli FH and mammalian aconitase, but not E. coli aconitase and mammalian FH [62]. Two other 4Fe‐4S cluster‐containing hydro‐lyases of E. coli were shown to lack OAT activity [62], indicating that OAT activity is a special characteristic and not a common feature of this class of enzymes.…”

Stereochemistry of enzymatic water addition to C = C bonds

“…No deuterium was present in the (S)-malate or fumaric acid in the reaction mixtures incubated with fumarase A or fumarase C, while the deuterium was still present in the (S)-malate in the fraction to which no enzyme was added. Since fumarase C is known to specifically eliminate water from (S)-malate in anti-fashion (Scheme 6, red box) (Gawron and Fondy, 1959;Flint, 1993) and the stereochemical course for fumarase A was identical it also had to be anti. Thus both fumarases catalyse the anti-addition of water to fumaric acid to form (S)-malate (Englard, 1960;Gawron et al, 1961;Karsna, 1958).…”