The animal
fatty acid synthase is a multifunctional
protein with a subunit molecular weight of 260,000. We recently reported the expression and characterization of the
acyl carrier protein and thioesterase domains of the chicken
liver fatty acid synthase in Escherichia coli. In order to gain insight into the mechanism of action of the thioesterase domain, we have replaced the putative active site
serine 101 with
alanine and
cysteine and the conserved
histidine 274 with
alanine by site-directed mutagenesis. While both the Ser101----Ala and His274----Ala
mutant proteins were inactive, the Ser101----Cys mutant
enzyme (
thiol-thioesterase) retained considerable activity, but the properties of the
enzyme were changed from an active
serine esterase to an active
cysteine esterase, providing strong evidence for the role of Ser101 as the active site nucleophile. In order to further probe into the role of His274, a double mutant was constructed containing both the Ser101----Cys and the His274----Ala mutations. The double-
mutant protein was inactive and exhibited diminished reactivity of the Cys-SH to
iodoacetamide as compared to that of the Ser101----Cys-thioesterase, suggesting a role of His274 as a general base in withdrawing the
proton from the Cys-SH in the
thiol-thioesterase or Ser101 in the wild-type
enzyme. Incubation of the recombinant thioesterases with [1-14C]
palmitoyl-CoA resulted in the incorporation of [1-14C] palmitoyl into the
enzyme only in the double mutant, suggesting that Cys-SH of the double mutant is reactive enough to form the palmitoyl-S-
enzyme intermediate. This intermediate is not hydrolyzed because of the lack of His274, which is required for the attack of H2O on the acyl
enzyme. These results suggest that the catalytic mechanism of the thioesterases may be similar to that of the
serine proteases and lipases, which employ a
serine-
histidine-
aspartic acid catalytic triad as part of their catalytic mechanism.