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Characterization of the reaction mechanism for the XL-I form of bovine liver xenobiotic/medium-chain fatty acid:CoA ligase.

ABSTRACT: 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.

SUBMITTER: Vessey DA

PROVIDER: S-EPMC1221953 | biostudies-other | 2001 Jul

REPOSITORIES: biostudies-other

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Project description:Mitochondrial fatty-acid beta-oxidation (mFAO) plays a central role in mammalian energy metabolism. Multiple severe diseases are associated with defects in this pathway. Its kinetic structure is characterized by a complex wiring of which the functional implications have hardly been explored. Repetitive cycles of reversible reactions, each cycle shortening the fatty acid by two carbon atoms, evoke competition between intermediates of different chain lengths for a common set of 'promiscuous' enzymes (enzymes with activity towards multiple substrates). In our validated kinetic model of the pathway, substrate overload causes a steep and detrimental flux decline. Here, we unravel the underlying mechanism and the role of enzyme promiscuity in it. Comparison of alternative model versions elucidated the role of promiscuity of individual enzymes. Promiscuity of the last enzyme of the pathway, medium-chain ketoacyl-CoA thiolase (MCKAT), was both necessary and sufficient to elicit the flux decline. Subsequently, Metabolic Control Analysis revealed that MCKAT had insufficient capacity to cope with high substrate influx. Next, we quantified the internal metabolic regulation, revealing a vicious cycle around MCKAT. Upon substrate overload, MCKAT's ketoacyl-CoA substrates started to accumulate. The unfavourable equilibrium constant of the preceding enzyme, medium/short-chain hydroxyacyl-CoA dehydrogenase, worked as an amplifier, leading to accumulation of upstream CoA esters, including acyl-CoA esters. These acyl-CoA esters are at the same time products of MCKAT and inhibited its already low activity further. Finally, the accumulation of CoA esters led to a sequestration of free CoA. CoA being a cofactor for MCKAT, its sequestration limited the MCKAT activity even further, thus completing the vicious cycle. Since CoA is also a substrate for distant enzymes, it efficiently communicated the 'traffic jam' at MCKAT to the entire pathway. This novel mechanism provides a basis to explore the role of mFAO in disease and elucidate similar principles in other pathways of lipid metabolism.

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Characterization of the reaction mechanism for the XL-I form of bovine liver xenobiotic/medium-chain fatty acid:CoA ligase.

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