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Studies on medium-chain fatty acyl-coenzyme a synthetase. Enzyme fraction I: mechanism of reaction and allosteric properties.

ABSTRACT: 1. The mechanism of butyrate activation catalysed by an enzyme fraction derived from ox liver particles (fraction I; Bar-Tana, Rose & Shapiro, 1968) was studied by an analysis of the initial-velocity pattern of the overall reaction and found to conform to the Bi Uni Uni Bi Ping Pong model (Cleland, 1963a,b,c) in agreement with the reaction scheme proposed by Berg (1956). 2. A homotropic co-operative effect was exerted by CoA on fraction I, whereas ATP and AMP functioned as heterotropic co-operative ligands with respect to butyryl-AMP-dependent CoA disappearance. On the other hand, PP(i) and butyryl-CoA showed antagonistic heterotropic effects when tested under similar conditions. With respect to the overall reaction CoA and ATP could be shown to function as co-operative homotropic modifiers. 3. Two interchangeable conformational states of the enzyme are therefore presumed to exist, state R, having a higher affinity for CoA and ATP and thus preferentially catalysing butyryl-AMP-dependent CoA disappearance (partial reaction b), and state T, favoured by the presence of PP(i), catalysing the formation of ATP from butyryl-AMP and PP(i) (partial reaction a) with greater efficiency. 4. These findings serve to explain the opposite effects of ATP on the partial reactions, as well as the inhibition by CoA and ATP of ATP formation (reaction a) and by PP(i) of the butyryl-AMP-dependent CoA disappearance (reaction b) (Bar-Tana et al. 1968). 5. The possible analogy of these observations to amino acid-activating and other similar systems is discussed.

SUBMITTER: Bar-Tana J

PROVIDER: S-EPMC1186785 | biostudies-other | 1968 Sep

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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Studies on medium-chain fatty acyl-coenzyme a synthetase. Enzyme fraction I: mechanism of reaction and allosteric properties.

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