Characterization of the reaction mechanism for the XL-I form of bovine liver xenobiotic/medium-chain fatty acid:CoA ligase.
The XL-I form of xenobiotic/medium-chain fatty acid:CoA ligase was purified to apparent homogeneity from bovine liver mitochondria and used to determine the reaction mechanism. A tersubstrate kinetic analysis was conducted by varying the concentrations of ATP, benzoate and CoA in turn. Both ATP and benzoate gave parallel double-reciprocal plots against CoA, which indicates a Ping Pong mechanism, with either pyrophosphate or AMP leaving before the binding of CoA. Addition of pyrophosphate to the assays changed the plots from parallel to intersecting; addition of AMP did not. This indicates that pyrophosphate is the product that leaves before binding of CoA. Based on end-product inhibition studies, it was concluded that the reaction follows a Bi Uni Uni Bi Ping Pong mechanism, with ATP binding first, followed in order by benzoate binding, pyrophosphate release, CoA binding, benzoyl-CoA release and AMP release. A similar mechanism was obtained when the ligase was examined with butyrate as substrate. However, butyrate activation was characterized by a much higher affinity for CoA. This is attributed to steric factors resulting from the bulkier nature of the benzoate molecule. Also, with butyrate there is a bivalent cation activation distinct from that associated with binding to ATP. This activation by excess Mg(2+) results in non-linear plots of 1/v against 1/[ATP] for butyrate unless the concentrations of Mg(2+) and ATP are varied together.
Vessey
D A
DA
Liver Study Unit, Department of Veterans' Affairs Medical Center, 4150 Clement St., San Francisco, CA 94121, USA. vessey.donald_a@sanfrancisco.va.gov
Kelley
M
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D000214:Acyl Coenzyme A / Q000494:pharmacology; D000249:Adenosine Monophosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001565:Benzoates / Q000493:pharmacokinetics; D002417:Cattle; D003065:Coenzyme A / Q000378:metabolism; D003066:Coenzyme A Ligases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D011756:Diphosphates / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D015262:Xenobiotics / Q000493:pharmacokinetics*
D000214:Acyl Coenzyme A; D001565:Benzoates; D011756:Diphosphates; D004791:Enzyme Inhibitors; D015262:Xenobiotics; D000249:Adenosine Monophosphate; D000255:Adenosine Triphosphate; D003066:Coenzyme A Ligases; C107128:xenobiotic medium chain fatty acid - coenzyme A ligase; D003065:Coenzyme A