142 Chapter 5 Table 1 shows the concentrations of the metabolites at steady state. The concentration of acyl-CoA and acyl-carnitine species is clearly higher than those of the enoyl-CoAs and ketoacyl-CoAs for all the chain lengths. Moreover, the ketoacyl-CoA species are the least abundant ones, probably due to the high equilibrium constant of ACAA1 (2.5·105). Table 2 shows the fluxes through the different enzymes at steady state. The flux of the pathway is lower than that described in the literature28. Moreover, the flux is significantly lower than the Vmax of ACOX1. In part this can be explained by the fact that we include six rounds of β-oxidation with substrates ranging from C18 through C8. Thus, only one sixth of the total ACOX1 pool is available for each round. In contrast, in experimental settings, the time is often relatively short, hence only one or two rounds of β-oxidation are observed. Further reduction of the flux relative to the Vmax of ACOX1 may be caused by almost complete saturation of the enzyme in the model by the high concentration of C18-acylCoA. This would leave little catalytic capacity for subsequent rounds. This is reminiscent of the vulnerability towards substrate overloading that was previously reported for a mitochondrial kinetic model59. For future studies, we aim to incorporate the import and export of products and substrates, and/or reduce the concentration of C18-acylCoA, to assess if peroxisomal β-oxidation is also sensitive towards overloading. Metabolic Control Analysis To analyze how the pathway would respond to regulation of enzyme concentrations, we applied the theoretical framework of Metabolic Control Analysis29. Here, the Flux Control Coefficient (FCC) of a specific enzyme i (CJ/ i) expresses the relative change of the flux J in response to a small increase in rate vi of this enzyme. The FCC of an enzyme i on the steady-state flux J is mathematically defined as: Here p is a parameter that selectively affects vi. An FCC close to 0 indicates that the enzyme has little to no effect on the pathway’s flux, while an FFC close to 1 indicates a high control over the rate. The summation of all the control coefficients of the enzymes involved in the pathway equals to 129. In order to numerically calculate the FCC of the different enzymes, the Vmaxs were individually changed (±0.1% of their maximal velocity) and the change of flux of the pathway was computed (Figure 2a)
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