143 5 Establishing a peroxisomal β-oxidation computational kinetic model to understand the effects of amino-acid restriction ACOX1 was found to be the enzyme with the highest FCC (0.58). In essence, this is in agreement with the hypothesis proposed before, that ACOX1 is the rate limiting enzyme of the peroxisomal β-oxidation28. However, this proposal was only based on the similarity between the ACOX1 kinetics and the flux, not on a formal analysis of flux control. This modelling result now supports the hypothesis. At the same time, the fact that the FCC is lower than 1, suggests that ACOX1 may share flux control with another enzyme. Indeed, ACAA1 also exerted a high control on the pathway’s flux. Control by ACAA1 has not been described before. Nevertheless, it is in line with studies on mitochondrial β-oxidation, where both experimental and computational analysis showed that the mitochondrial ketoacyl-CoA thiolase (MCKAT) took over some control under specific conditions59,60. The concentration of the C18-acylCoA substrate (10 μM versus 150 μM) had only a minor effect on the distribution of flux control (Figure 2a). Typically, flux control tends to decrease if an enzyme is activated. Indeed, when increasing the Vmax of ACOX1 by a factor of five, a shift in control towards ACAA1 was observed (Figure 2b). This indicates that the distribution of flux control may be regulated by gene expression and enzyme content. It also underlines the relevance of reliable kinetic parameters. As mentioned in the literature review part of this paper, very disparate parameters were found for the Vmax of Catalase38,61,62. It has been reported that the Km and the Vmax of catalase are extremely high. Since we could not find kinetic parameters for catalase isolated from human or rodent liver, we used the average of different parameters for catalases isolated from different origins. These values were extremely high, with a calculated Vmaxhigher than 46000 µmol ·min-1·mg PeroxisomalProt-1 and a K m of 93000 µM38. Interestingly, another study63 measured the V max of catalase in human liver tissue and reported it to be 0.18 µmol · min-1·mg liver Prot-1. Upon conversion to peroxisomal protein (see calculations above), this becomes 9.375 µmol · min-1·mg peroxisomal Prot-1, hence much lower than the above-mentioned values. However, the control coefficient distribution remained the same with the higher and the lover Vmax values. The flux of the pathway only changed minimally. Given that the lower value was closer to those of the rest of the enzymes, all the predictions were performed using this Vmax value. Finally, we also modeled the catalase rate with the reported second order rate constant k = 7.9·106 M-1 s-1 41. To do so, the rate of the enzyme was defined by:
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