PMID:1684105
| Citation |
Man, WJ, Li, Y, O'Connor, CD and Wilton, DC (1991) Conversion of citrate synthase into citryl-CoA lyase as a result of mutation of the active-site aspartic acid residue to glutamic acid. Biochem. J. 280 ( Pt 2):521-6 |
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| Abstract |
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]. |
| Links | |
| Keywords |
Acetyl Coenzyme A/metabolism; Aspartic Acid/genetics; Base Sequence; Binding Sites; Catalysis; Citrate (si)-Synthase/genetics; Citrate (si)-Synthase/metabolism; DNA; Escherichia coli/enzymology; Glutamates/genetics; Glutamic Acid; Lyases/metabolism; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutation |
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