نبذة مختصرة : 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.
Author summary Burning of fat is essential to provide energy to our bodies. However, fat metabolism is also involved in well-known diseases, such as metabolic syndrome and type 2 diabetes. In severe, inherited deficiencies of enzymes in the fatty-acid oxidation pathway, children run the risk of life-threatening low glucose levels or ‘hypoglycemia’. This typically occurs during overnight fasting, when the body depends on fat stores for its energy supply. To understand the sequence of events leading to hypoglycemia and eventually provide novel therapies, we simulated the process with a validated computer model. We found that when the supply of fat to the liver was increased beyond a critical value, the rate of fatty-acid oxidation declined. Under these circumstances, intermediate metabolites accumulated, very much like the situation in a traffic jam. The risk of a metabolic traffic jam was enhanced in fatty-acid oxidation disorders. In the body this would lead to insufficient energy to maintain blood glucose levels. In this paper we unravel the vicious cycle that leads to the detrimental flux decline and identify a critical role for the last enzyme of the pathway, MCKAT. Our results suggests that MCKAT or its regulators may be promising drug targets.
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