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J. Biol. Chem., Vol. 277, Issue 49, 47270-47275, December 6, 2002
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From the Human bile acid-CoA:amino acid
N-acyltransferase (hBAT), an enzyme catalyzing the
conjugation of bile acids with the amino acids glycine or taurine has
significant sequence homology with dienelactone hydrolases and other
Conserved Residues in the Putative Catalytic Triad of Human Bile
Acid Coenzyme A:Amino Acid N-Acyltransferase*
,
,, and§**
Department of Pharmacology and Toxicology,
the ¶ Department of Microbiology, and the
§ Comprehensive Cancer Center Mass Spectrometry Shared
Facility, University of Alabama at Birmingham,
Birmingham, Alabama 35294
/
hydrolases. These enzymes have a conserved catalytic triad that
maps onto the mammalian BATs at residues Cys-235, Asp-328, and His-362
of the human sequence, albeit that the hydrolases contain a serine
instead of a cysteine. In the present study, the function of the
putative catalytic triad of hBAT was examined by chemical modification
with the cysteine alkylating reagent N-ethylmaleimide (NEM)
and by site-directed mutagenesis of the triad residues followed by
enzymology studies of mutant and wild-type hBATs. Treatment with NEM
caused inactivation of wild-type hBAT. However, preincubation of
wild-type hBAT with the substrate cholyl-CoA before NEM treatment
prevented loss of N-acyltransferase activity. Substitution
of His-362 or Asp-328 with alanine results in inactivation of hBAT.
Although substitution of Cys-235 with serine generated an hBAT mutant
with lower N-acyltransferase activity, it substantially
increased the bile acid-CoA thioesterase activity compared with wild
type. In summary, data from this study support the existence of an
essential catalytic triad within hBAT consisting of Cys-235, His-362,
and Asp-328 with Cys-235 serving as the probable nucleophile and thus
the site of covalent attachment of the bile acid molecule.
*
This work was supported by National Institutes of Health
Grants DK-46390 (to S. B.) and AI43230 (to M. S.) The mass
spectrometer was purchased by funds from National Institutes of Health
Instrumentation Grant (S10RR06487) and from this institution.
Operation of the Mass Spectrometry and Oligonucleotide Shared
Facilities has been supported in part by a NCI National Institutes of
Health Core Research Support Grant P30 CA13148 to the University of
Alabama at Birmingham Comprehensive Cancer Center.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Current address: Dept. of Microbiology and Immunology,
University of Oklahoma Health Sciences Center, Oklahoma City, OK 73190.
**
To whom correspondence should be addressed. Tel.: 205-934-7117;
Fax: 205-934-6944; E-mail: sbarnes@uab.edu.
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