137 5 Establishing a peroxisomal β-oxidation computational kinetic model to understand the effects of amino-acid restriction (SCPx). Human peroxisomes contain two thiolases: the peroxisomal 3-ketoacylCoA thiolase (ACAA1) and the Sterol Carrier Protein x (SCP2)2. Here, we focus on the role of 3-ketoacyl-CoA thiolase (ACAA1) in the metabolism of straight-chain fatty acids. Cleland et al48 proposed that peroxisomal 3-ketoacyl-CoA thiolase followed a ping-pong mechanism. This was later confirmed by Miyazawa et al49. In 1981 Miyazawa et al. isolated the enzyme from rat liver and compared its kinetic properties to the mitochondrial thiolases49. Interestingly, at this point only one peroxisomal 3-ketoacyl-CoA thiolase was reported and it was not specified whether it was A or B. They reported low Km values for the longer-chain substrates and the highest velocity with 3-ketooctanoyl-CoA as the substrate. In later studies, the measured activities and substrate specificities of the 3-ketyoacyl-CoA thiolases A and B from livers of rats treated with clofibrate were the same 50. Even though the reaction catalyzed by peroxisomal 3-ketoacyl-CoA thiolase is in principle reversible, the thiolysis reaction is thermodynamically favoured over the condensation reation51,47. The enzyme can be inhibited by both CoA and acetyl-CoA. Substrate inhibition by CoA is, however, only observed at concentrations higher than 100 µM. 49 Carnitine acetyltransferase and carnitine octanoyltransferase Once the acyl-CoAs have been shortened, they can either enter into a new cycle of β-oxidation or be converted to their cognate carnitine ester by one of two enzymes, namely carnitine acetyltransferase (CRAT) or carnitine octanoyltransferase (CROT). Acylcarnitines are exported out of the peroxisomes and transported into mitochondria via the carnitine acylcarnitine carrier52. In 1983, Miyazawa et al purified and characterized both carnitine octanoyltransferase and carnitine acetyltransferase from rat liver 53,54. When assaying CROT, they reported higher Km values for shorter chain-lengths (C2-C6) compared to the low and constant values for substrates between C8 and C18. However, Farrel et al.55 reported that the apparent K m values for the murine enzyme were not constant: these values were smaller for substrates between C4 and C12 and larger for C14, C16 and C18. Both groups reported CROT to exhibit its maximum activity towards hexanoyl-CoA, while the maximum activity of CRAT was observed towards butyryl-CoA55, highlighting the difference in chain-length specificity between the two enzymes. CRAT exhibited very similar values for the assayed acyl-CoAs (C2-C8) with a higher value for C12. Farrel et al. also reported that the chain length of the acyl-CoA influenced the Km for L-carnitine. This is highly important in vivo, given the
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