Bile acids (BAs) function in the biliary excretion of non-polar compounds and the intestinal absorption of lipids, but only if they are conjugated with glycine (GLY) or taurine (tau). Also, the excretion of BAs into bile has been shown to require prior conjugation. Thus factors affecting the rate of conjugation of each individual BAs need to be understood. This requires knowledge of the enzymology of BA conjugation. Conjugation involves an initial activation of the BA to bile acid-CoA in a reaction catalyzed by microsomal Bile Acid:CoA Ligase and then conjugation with gly or tau in a reaction catalyzed by gly/tau N-acyltransferase (N-ATase). The ligase has been found to be rate limiting for the conjugation of the 4 common BAs; this needs to be determined for other BAs. Ligase activity in vivo is expected to be sensitive to factors which affect the cellular concentration of the substrate CoA and this will be studied. The active site chemistry of the ligase will be probed by determining the reaction mechanism and the role of Mn2+. We can then determine the enzyme's substrate specificity which is important as it affects the ratio of mono- to di- to tri-hydroxy BAs in bile and thereby also cholesterol solubility. To aid in correlating the structure of the ligase to its function, the amino acid sequence for the ligase will be determined. The sequence will also: help orient active site labeling probes, and reveal potential membrane spanning segments and disclose signal sequences directing membrane insertion. The ligase/membrane topography will be mapped. For the N- ATase, the ratio of gly to tau conjugation varies with the structure of the BA. This is significant as gly conjugation favors cholesterol solubility in bile. To understand the basis for the gly/tau ratio, we will look for factors which affect catalysis. The enzyme's amino acid sequence will be obtained. then the known covalent intermediate between BA and the enzyme active site will be studied to determine the nature and location of the amino acid residue to which the BA is bound. We will then use site- directed mutagenesis to examine the exact role of this residue and other critical residues. The role of the CoA moiety in promoting catalysis will be determined. To explain the failure of non-mammalian forms of N-ATase to conjugate with gly, we will determine the amino acid sequence of the N- ATase from chicken liver for comparison with the bovine form. The glucuronidation of BAs becomes important during cholestasis and we will study the regulation of the glucuronyltransferases using radiation inactivation analysis.
