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

J Biol Chem. 2002 Dec 6;277(49):47270-5. doi: 10.1074/jbc.M207463200. Epub 2002 Sep 17.

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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

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

Substances

  • Acyl Coenzyme A
  • Bile Acids and Salts
  • Cholic Acids
  • DNA, Complementary
  • cholyl-coenzyme A
  • Aspartic Acid
  • Histidine
  • Acyltransferases
  • bile acid-CoA amino acid N-acyltransferase
  • Trypsin
  • Cysteine
  • Ethylmaleimide
  • Coenzyme A