Document Detail


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.
MedLine Citation:
PMID:  8446599     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
M H Tai; S S Chirala; S J Wakil
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  Proceedings of the National Academy of Sciences of the United States of America     Volume:  90     ISSN:  0027-8424     ISO Abbreviation:  Proc. Natl. Acad. Sci. U.S.A.     Publication Date:  1993 Mar 
Date Detail:
Created Date:  1993-04-08     Completed Date:  1993-04-08     Revised Date:  2009-11-18    
Medline Journal Info:
Nlm Unique ID:  7505876     Medline TA:  Proc Natl Acad Sci U S A     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  1852-6     Citation Subset:  IM    
Affiliation:
Verna and McLean Department of Biochemistry, Baylor College of Medicine, Houston, TX 77030.
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MeSH Terms
Descriptor/Qualifier:
Amino Acid Sequence
Animals
Base Sequence
Binding Sites
Cloning, Molecular
Diethyl Pyrocarbonate / pharmacology
Fatty Acid Synthetase Complex / antagonists & inhibitors,  chemistry*,  metabolism*
Molecular Sequence Data
Mutagenesis, Site-Directed
Palmitoyl Coenzyme A / metabolism
Phenylmethylsulfonyl Fluoride / pharmacology
Rats
Recombinant Proteins / metabolism
Structure-Activity Relationship
Thiolester Hydrolases / antagonists & inhibitors,  chemistry*,  metabolism
Grant Support
ID/Acronym/Agency:
GM-19091/GM/NIGMS NIH HHS
Chemical
Reg. No./Substance:
0/Recombinant Proteins; 1609-47-8/Diethyl Pyrocarbonate; 1763-10-6/Palmitoyl Coenzyme A; 329-98-6/Phenylmethylsulfonyl Fluoride; EC 3.1.2.-/Thiolester Hydrolases; EC 3.1.2.-/thioesterase II; EC 6.-/Fatty Acid Synthetase Complex
Comments/Corrections

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