INITIAL KINETIC AND MECHANISTIC CHARACTERIZATION OF ESCHERICHIA-COLI FUMARASE-A

The protein encoded by the fumA gene in Escherichia coli is shown here in to be a highly efficient and specific catalyst of the fumarase reac tion. In an investigation of 21 substrate analogs, this protein only h ad substantial activity as a hydro-lyase on fumarate, malate, acetylen e dicarboxylate, fluorofumarate, and 2(S),3(S)-tartrate. The k(cat) an d k(cat)/K-m 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 p hysiological role of this protein is a catalyst of the fumarase reacti on; therefore, it is appropriate to name it fumarase A. Fumarase A spe cifically removes the 3-pro-R in the dehydration of (2S)-malate. The p roduct of the action of fumarase A on acetylene dicarboxylate, fluorof umarate and 2(S),3(S)-tartrate is oxalacetate. The nitronate form of 2 -hydroxy-3-nitro-propionate is a potent inhibitor of fumarase A, imply ing that the enzyme forms an intermediate with an anion at C-3. No kin etic isotope effect was found with (2S,3R)-3-[H-2]malate. The effects of pH on the k(cat) and k(cat)/K-m for fumarate as a substrate show th at the pK(a)s of the groups involved in catalysis differ markedly from porcine fumarase. The possible roles of the proteins encoded by the t hree fumarase genes in E. coli are briefly discussed. (C) 1994 Academi c Press, Inc.