Site-directed mutagenesis of citrate synthase; the role of the active-site aspartate in the binding of acetyl-CoA but not oxaloacetate.
Asp-362, a potential key catalytic residue of Escherichia coli citrate synthase (citrate oxaloacetate-lyase [pro-3S)-CH2COO- ----acetyl-CoA), EC 4.1.3.7) has been converted to Gly-362 by oligonucleotide-directed mutagenesis. The mutant gene was completely sequenced, using a series of synthetic oligodeoxynucleotides spanning the structural gene to confirm that no additional mutations had occurred during genetic manipulation. The mutant gene was expressed in M13 bacteriophage and produced a protein which migrated in an identical manner to wild-type E. coli citrate synthase on SDS-polyacrylamide gels and which cross-reacted with E. coli citrate synthase antiserum. The mutant gene was subsequently recloned into pBR322 for large scale purification of the protein, and the resulting plasmid, pCS31, used to transform the citrate synthase deletion strain, W620. The mutant enzyme purified in an analogous manner to wild-type E. coli citrate synthase and expressed less than 2% of wild-type enzyme activity. The activity of the partial reactions catalysed by citrate synthase was similarly affected suggesting that this residual activity may be due to contaminating wild-type enzyme activity. The mutant citrate synthase retains a high-affinity NADH-binding site consistent with the protein preserving its overall structural integrity. Oxaloacetate binding to the protein is unaffected by the Asp-362 to Gly-362 mutation. Binding of the acetyl-CoA analogue, carboxymethyl-CoA, could not be detected in the mutant protein indicating that the lack of catalytic competence is due primarily to the inability of the protein to bind the second substrate, acetyl-CoA.
Handford
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PA
Department of Biochemistry, University of Southampton, U.K.
Ner
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Bloxham
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DP
Wilton
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DC
D000105:Acetyl Coenzyme A / Q000378:metabolism*; D001224:Aspartic Acid; D001435:Bacteriophages / Q000201:enzymology; D001483:Base Sequence; D001665:Binding Sites; D002950:Citrate (si)-Synthase / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007118:Immunoassay; D009154:Mutation; D009243:NAD / Q000378:metabolism; D010071:Oxaloacetates / Q000378:metabolism*; D007652:Oxo-Acid-Lyases / Q000378:metabolism*; D010957:Plasmids; D013329:Structure-Activity Relationship; D014169:Transformation, Bacterial
D004274:DNA, Recombinant; D010071:Oxaloacetates; D009243:NAD; D001224:Aspartic Acid; D000105:Acetyl Coenzyme A; D002950:Citrate (si)-Synthase; D007652:Oxo-Acid-Lyases