Biosynthetic thiolase from Zoogloea ramigera. Mutagenesis of the putative active-site base Cys-378 to Ser-378 changes the partitioning of the acetyl S-enzyme intermediate

J Biol Chem. 1992 Aug 15;267(23):16041-3.


The proposed active-site base Cys-378 of thiolase, responsible for deprotonation of acetyl-CoA, has been converted to a less acidic residue Ser-378 by mutagenesis. Comparison of the CD spectra and dimethyl suberimidate cross-linking experiments of the wild type, mutant Ser-378, and Gly-378 enzymes indicated that there have been no major conformational changes. The Ser-378 enzyme retains 0.1% of the Vmax of wild type in the direction of acetoacetyl-CoA thiolytic cleavage and 0.07% of the Vmax in the Claisen condensation direction. Analysis of the acetyl S-enzyme intermediate partitioning, that is capture of the acetyl enzyme by 1) the thiolate of coenzyme A relative to 2) the C-2 carbanion of acetyl-CoA, is changed to favor reaction 2 in the case of the Ser-378 mutant enzyme.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Acetyl-CoA C-Acetyltransferase / biosynthesis
  • Acetyl-CoA C-Acetyltransferase / genetics
  • Acetyl-CoA C-Acetyltransferase / metabolism*
  • Acetylation
  • Amino Acid Sequence
  • Binding Sites
  • Circular Dichroism
  • Cysteine / genetics*
  • Kinetics
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed*
  • Protein Conformation
  • Pseudomonadaceae / enzymology*
  • Serine


  • Serine
  • Acetyl-CoA C-Acetyltransferase
  • Cysteine

Associated data

  • GENBANK/J02631
  • GENBANK/J02636
  • GENBANK/M94136
  • GENBANK/M94137
  • GENBANK/M94138
  • GENBANK/M94139
  • GENBANK/M94140
  • GENBANK/M94141
  • GENBANK/M94142
  • GENBANK/M94143
  • GENBANK/M94144