Initial kinetic and mechanistic characterization of Escherichia coli fumarase A.
The protein encoded by the fumA gene in Escherichia coli is shown herein to be a highly efficient and specific catalyst of the fumarase reaction. In an investigation of 21 substrate analogs, this protein only had substantial activity as a hydro-lyase on fumarate, malate, acetylene dicarboxylate, fluorofumarate, and 2(S),3(S)-tartrate. The kcat and kcat/Km for the hydration of fumarate by this protein are 3100 s-1 and 5 x 10(6) mol-1 s-1, respectively. It is likely that one physiological role of this protein is a catalyst of the fumarase reaction; therefore, it is appropriate to name it fumarase A. Fumarase A specifically removes the 3-pro-R in the dehydration of (2S)-malate. The product of the action of fumarase A on acetylene dicarboxylate, fluorofumarate and 2(S),3(S)-tartrate is oxalacetate. The nitronate form of 2-hydroxy-3-nitro-propionate is a potent inhibitor of fumarase A, implying that the enzyme forms an intermediate with an anion at C-3. No kinetic isotope effect was found with (2S,3R)-3-[2H]malate. The effects of pH on the kcat and kcat/Km for fumarate as a substrate show that the pKas of the groups involved in catalysis differ markedly from porcine fumarase. The possible roles of the proteins encoded by the three fumarase genes in E. coli are briefly discussed.
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E. I. du Pont de Nemours and Co., Wilmington, Delaware 19880.
D000818:Animals; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005650:Fumarates / Q000378:metabolism; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D019746:Intramolecular Oxidoreductases; D007535:Isomerases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008293:Malates / Q000378:metabolism; D015394:Molecular Structure; D013379:Substrate Specificity; D013552:Swine
D005650:Fumarates; D008293:Malates; D007535:Isomerases; D019746:Intramolecular Oxidoreductases; C048474:oxaloacetate tautomerase