Determination of the mechanism of reaction for bile acid: CoA ligase.
The reaction of cholic acid, CoA and ATP to yield cholyl-CoA was investigated by kinetic analysis of the reaction as catalysed by guinea pig liver microsomes. The enzyme has an absolute requirement for divalent cation for activity so all kinetic analyses were carried out in excess Mn2+. A trisubstrate kinetic analysis was conducted by varying, one at a time ATP cholate and CoA. Both ATP and cholate gave parallel double reciprocal plots versus CoA, which indicates a ping-pong mechanism with either pyrophosphate or AMP leaving prior to the binding of CoA. Addition of pyrophosphate to the assays changed the parallel plots to intersecting ones; addition of AMP did not. This indicates that pyrophosphate is the first product. The end-product, AMP, was a competitive inhibitor versus ATP, as was cholyl-CoA at saturating concentrations of cholate. Both AMP and cholyl-CoA were uncompetitive inhibitors versus CoA. Based on this information, it was concluded that the reaction follows a bi uni uni bi ping-pong mechanism with ATP binding first, and with the release of the final products, AMP and cholyl-CoA, being random. CoA showed substrate inhibition at high but non-saturating concentrations and this inhibition was competitive versus ATP, which is consistent with the predicted ping-pong mechanism. The ability of cholyl-CoA, but not cholate or CoA, to bind with high affinity to the free enzyme was suggestive of a high affinity of the enzyme for the thioester link.
Kelley
M
M
Liver Study Unit, Department of Veterans' Affairs Medical Center, San Francisco, CA 94121.
Vessey
D A
DA
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D003066:Coenzyme A Ligases / Q000378:metabolism*; D011756:Diphosphates / Q000378:metabolism; D006168:Guinea Pigs; D006859:Hydrogen / Q000378:metabolism; D006878:Hydroxides / Q000378:metabolism; D007700:Kinetics; D008862:Microsomes, Liver / Q000201:enzymology
D011756:Diphosphates; D006878:Hydroxides; D006859:Hydrogen; D000255:Adenosine Triphosphate; D003066:Coenzyme A Ligases; C070656:bile acid-CoA ligase