Roles of Ser101, Asp236, and His237 in catalysis of thioesterase II and of the C-terminal region of the enzyme in its interaction with fatty acid synthase.
Thioesterase II (TE II), present in specialized tissues, catalyzes the chain termination and release of medium-chain fatty acids from fatty acid synthase [FAS; acyl-CoA:malonyl-CoA C-acyltransferase (decarboxylating, oxoacyl- and enoyl-reducing and thioester-hydrolyzing), EC 2.3.1.85]. We have expressed rat mammary gland TE II in Escherichia coli and created several site-directed mutants. Replacing both Ser101 and His237 with Ala yielded inactive proteins, suggesting that these residues are part of the catalytic triad as in FAS thioesterase (TE I). Mutating the conserved Asp236 or modifying it with Woodward's reagent K caused partial loss (40%) of TE II activity and reduced reactivity of Ser101 and His237 toward their specific inhibitors, phenylmethylsulfonyl fluoride and diethylpyrocarbonate, respectively. These results suggested that Asp236 enhances, but is not essential for, the reactivity of Ser101 and His237. Mutation analyses revealed that, at the C terminus, Leu262 is critical for TE II to interact with FAS. Hydrophobic interactions seem to play a role, since the interaction of TE II with FAS is enhanced by polyethylene glycol but reduced by salt. The Ser101 and His237 mutants and a synthetic C-terminal decapeptide did not compete in the interaction. These results suggest that a TE II-acyl FAS complex forms first, which then is stabilized by the interaction of the hydrophobic C terminus of TE II with FAS, leading ultimately to hydrolysis and release of fatty acid.
Tai
M H
MH
Verna and McLean Department of Biochemistry, Baylor College of Medicine, Houston, TX 77030.
Chirala
S S
SS
Wakil
S J
SJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D064429:Fatty Acid Synthases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010171:Palmitoyl Coenzyme A / Q000378:metabolism; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D013869:Thiolester Hydrolases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism
D011994:Recombinant Proteins; D010171:Palmitoyl Coenzyme A; D010664:Phenylmethylsulfonyl Fluoride; D064429:Fatty Acid Synthases; D013869:Thiolester Hydrolases; C022738:thioesterase II; D004047:Diethyl Pyrocarbonate