Conserved residues in the putative catalytic triad of human bile acid Coenzyme A:amino acid N-acyltransferase.

Academic Article

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.
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    Keywords

  • Acyl Coenzyme A, Acyltransferases, Amino Acid Sequence, Aspartic Acid, Bile Acids and Salts, Catalysis, Catalytic Domain, Cholic Acids, Coenzyme A, Conserved Sequence, Cysteine, DNA, Complementary, Dose-Response Relationship, Drug, Ethylmaleimide, Histidine, Humans, Kinetics, Models, Chemical, Molecular Sequence Data, Mutation, Protein Binding, Sequence Homology, Amino Acid, Spectrometry, Mass, Electrospray Ionization, Time Factors, Trypsin
  • Digital Object Identifier (doi)

    Author List

  • Sfakianos MK; Wilson L; Sakalian M; Falany CN; Barnes S
  • Start Page

  • 47270
  • End Page

  • 47275
  • Volume

  • 277
  • Issue

  • 49