The promiscuous enzyme medium-chain 3-keto-acyl-CoA thiolase triggers a vicious cycle in fatty-acid beta-oxidation

Abstract

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.

Multiple acyl-CoA thioesterases occupy distinct functional niches within the mitochondrial matrix

“…Alternatively, different matrix Acots could occupy distinct functional niches, defined by tissue specificity and/or regulation. These are important questions because matrix Acots are poised to regulate mitochondrial β-oxidation which is susceptible to overload with consequences on β-oxidation itself (5,34), on other matrix pathways and ultimately on other aspects of cell function. Thus understanding the characteristics of Acots, both individually and as a group, should provide insight into how Acots influence βoxidation, and also how best to study the matrix Acots.…”

“…An ability of matrix Acots to compete with β-oxidation implies that matrix processes can exert control on β-oxidation, a concept that conflicts with the generally accepted model that the main control point for β-oxidation is the entry of activated long-chain fatty acids into mitochondria at CPT1 (carnitine palmitoyl-transferase-1) at the OMM. However, recent experimental and modeling studies show that control of β-oxidation can be partly or substantially taken over by the β-oxidation reactions themselves (5,34). This shift in control occurred at high concentrations of long-chain fatty acyl-CoA and lead to the accumulation of intermediates and partial depletion of CoA (34).…”

“…The possibility that β-oxidation enzymes exert control over β-oxidation was also suggested by measurements of β-oxidation intermediates (for review: (45)). More recent modeling studies indicate that, at high C16:0-CoA concentration, control of β-oxidation shifts to the C4-C6 reactions of MCKAT (medium-chain ketoacyl-CoA thiolase) (5). This potential for overload, which could theoretically halt β-oxidation (5), suggests the utility of the Acots to mitigate gridlock at MCKAT and to prevent CoA depletion.…”

“…Models of β-oxidation do not include Acot reactions (5,34). The present study facilitates the incorporation of Acots into these, which could help to shed further light on the extent of the influence of Acots on β-oxidation, including in conditions of potential substrate overload.…”

“…Acots are predicted to have a high biological relevance because their substrates, acyl-CoA esters, are also substrates for other enzymes that serve major metabolic pathways, such as mitochondrial β-oxidation. Interestingly, β-oxidation overload has been described in the context of high substrate supply, and the bottleneck was localized to the mitochondrial matrix (5) raising the question of a role for Acots to mitigate the overload. Beyond β-oxidation, acyl-CoA esters can serve as allosteric or covalent regulators, which greatly expands the potential biological relevance of thioesterases.…”