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.