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.
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.
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
S F
SF
Williams
M A
MA
Palmer
O P
OP
Peoples
C T
CT
Walsh
A J
AJ
Sinskey
S
S
Masamune
D000101:Acetyl-CoA C-Acetyltransferase / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D000107:Acetylation; D000595:Amino Acid Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D003545:Cysteine / Q000235:genetics*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011548:Pseudomonadaceae / Q000201:enzymology*; D012694:Serine
D012694:Serine; D000101:Acetyl-CoA C-Acetyltransferase; D003545:Cysteine