2-Fluoro-beta-alanine, a previously unrecognized substrate for bile acid coenzyme A:amino acid:N-acyltransferase from human liver.
Our laboratory has demonstrated recently that conjugates of 2-fluoro-beta-alanine (FBAL) and bile acids are the major biliary metabolites of 5-fluorouracil (FUra) in cancer patients. Bile acids are normally conjugated with glycine or taurine, and therefore the identification of the FBAL-bile acid conjugates suggested that FBAL may also be a substrate for the bile acid conjugating enzyme, bile acid CoA:amino acid:N-acyltransferase. Enzyme activity detected using glycine and taurine as substrates was purified 8-fold from human liver cytosol using a DEAE-cellulose column. This preparation when tested for its activity towards beta-alanine and FBAL using cholyl CoA as the bile acid substrate only catalyzed the formation of FBAL-cholate. beta-Alanine was not a substrate. Confirmation of FBAL-cholate as the enzymatic product was demonstrated by (1) coelution of the product of this reaction on HPLC with authentic FBAL-cholate, (2) specific hydrolysis of this product by cholylglycine hydrolase, and (3) molecular weight of the product (497) being identical to that of the authentic FBAL-cholate. Kinetic experiments demonstrated that the enzyme had an affinity for FBAL (Km 1.45 mM) comparable to taurine (Km 1.32 mM), but greater than glycine (Km 6.45 mM). Formation of FBAL-cholate was inhibited competitively by taurine (Ki 1.27 mM) and glycine (Ki 4.47 mM), suggesting that a single enzyme is responsible for conjugation of glycine, taurine and FBAL with bile acids. These data indicate that the formation of the FBAL-bile acid conjugates in patients receiving FUra results from high affinity of the bile acid conjugating enzyme for FBAL.
Johnson
M R
MR
Department of Pharmacology, University of Alabama, Birmingham 35294.
Barnes
S
S
Sweeny
D J
DJ
Diasio
R B
RB
D000217:Acyltransferases / Q000378:metabolism*; D000409:Alanine / Q000031:analogs & derivatives*; D000581:Amidohydrolases / Q000378:metabolism; D001647:Bile Acids and Salts / Q000378:metabolism*; D002793:Cholic Acids / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D046508:Culture Techniques; D005998:Glycine / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D013379:Substrate Specificity; D013654:Taurine / Q000494:pharmacology; D015091:beta-Alanine / Q000031:analogs & derivatives* / Q000032:analysis / Q000378:metabolism
D001647:Bile Acids and Salts; D002793:Cholic Acids; C052999:N-cholyl-2-fluoro-beta-alanine; D015091:beta-Alanine; D013654:Taurine; C032348:alpha-fluoro-beta-alanine; D000217:Acyltransferases; C025118:cholyl CoA glycine-taurine N-acyltransferase; D000581:Amidohydrolases; C023154:choloylglycine hydrolase; D000409:Alanine; D005998:Glycine