title: Conserved residues in the putative catalytic triad of human bile acid Coenzyme A:amino acid N-acyltransferase.
abstract: 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 alpha/beta 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.
authors.affiliation: Department of Pharmacology and Toxicology, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.
authors.forename: Mindan K
authors.initials: MK
authors.lastname: Sfakianos
authors.forename: Landon
authors.initials: L
authors.lastname: Wilson
authors.forename: Michael
authors.initials: M
authors.lastname: Sakalian
authors.forename: Charles N
authors.initials: CN
authors.lastname: Falany
authors.forename: Stephen
authors.initials: S
authors.lastname: Barnes
mesh_terms: D000214:Acyl Coenzyme A / Q000378:metabolism; D000217:Acyltransferases / Q000737:chemistry*; D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000737:chemistry; D001647:Bile Acids and Salts / Q000378:metabolism*; D002384:Catalysis; D020134:Catalytic Domain; D002793:Cholic Acids / Q000378:metabolism; D003065:Coenzyme A / Q000737:chemistry*; D017124:Conserved Sequence; D003545:Cysteine / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005033:Ethylmaleimide / Q000494:pharmacology; D006639:Histidine / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D021241:Spectrometry, Mass, Electrospray Ionization; D013997:Time Factors; D014357:Trypsin / Q000494:pharmacology
chemical_list: D000214:Acyl Coenzyme A; D001647:Bile Acids and Salts; D002793:Cholic Acids; D018076:DNA, Complementary; C117585:cholyl-coenzyme A; D001224:Aspartic Acid; D006639:Histidine; D000217:Acyltransferases; C026953:bile acid-CoA amino acid N-acyltransferase; D014357:Trypsin; D003545:Cysteine; D005033:Ethylmaleimide; D003065:Coenzyme A