Conversion of citrate synthase into citryl-CoA lyase as a result of mutation of the active-site aspartic acid residue to glutamic acid.
The active-site aspartic acid residue, Asp-362, of Escherichia coli citrate synthase was changed by site-directed mutagenesis to Glu-362, Asn-362 or Gly-362. Only very low catalytic activity could be detected with the Asp----Asn and Asp----Gly mutations. The Asp----Glu mutation produced an enzyme that expressed about 0.8% of the overall catalytic rate, and the hydrolysis step in the reaction, monitored as citryl-CoA hydrolysis, was inhibited to a similar extent. However, the condensation reaction, measured in the reverse direction as citryl-CoA cleavage to oxaloacetate and acetyl-CoA, was not affected by the mutation, and this citryl-CoA lyase activity was the major catalytic activity of the mutant enzyme. This high condensation activity in an enzyme in which the subsequent hydrolysis step was about 98% inhibited permitted considerable exchange of the methyl protons of acetyl-CoA during catalysis by the mutant enzyme. The Km for oxaloacetate was not significantly altered in the D362E mutant enzyme, whereas the Km for acetyl-CoA was about 5 times lower. A mechanism is proposed in which Asp-362 is involved in the hydrolysis reaction of this enzyme, and not as a base in the deprotonation of acetyl-CoA as recently suggested by others. [Karpusas, Branchaud & Remington (1990) Biochemistry 29, 2213-2219; Alter, Casazza, Zhi, Nemeth, Srere & Evans, (1990) Biochemistry 29, 7557-7563].
Department of Biochemistry, University of Southampton, Bassett Crescent East, U.K.
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Man
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O'Connor
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Wilton
D000105:Acetyl Coenzyme A / Q000378:metabolism; D001224:Aspartic Acid / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D002950:Citrate (si)-Synthase / Q000235:genetics* / Q000378:metabolism; D004247:DNA; D004926:Escherichia coli / Q000201:enzymology; D005971:Glutamates / Q000235:genetics*; D018698:Glutamic Acid; D008190:Lyases / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation
D005971:Glutamates; D001224:Aspartic Acid; D018698:Glutamic Acid; D000105:Acetyl Coenzyme A; D004247:DNA; D002950:Citrate (si)-Synthase; D008190:Lyases