140 Chapter 5 The computational model consists of 6 enzymes with their corresponding rate equations, 24 ordinary differential equations (ODE) for variable metabolite concentrations, and additionally 7 boundary fixed metabolite concentrations (for a detailed equations see Supplementary Text 1). ODEs describe the change of different metabolites in time as a result of the enzyme rates (production and consumption). Model simulations allow to predict time courses of all the different variable metabolite concentrations in the pathway, including acyl-CoAs, enoyl-CoAs, ketoacyl-CoAs and acylcarnitines of the different chain lengths (C18 to C8). Subsequently, concentrations of the different metabolites as well as the enzyme fluxes are calculated at the steady state. Assumptions and conversion factors Vmax values are given in µmol of product per minute per milligram of peroxisomal protein. For the calculation of Vmax values, the following scenarios were encountered. (i) When the activity was measured in purified enzyme, the Vmax was corrected by the concentration of the enzyme in a human liver47 and then converted to peroxisomal protein, using that 1.92% of the liver protein content is peroxisomal protein57. (ii) When the activity of the enzyme was measured in the peroxisomal fraction and expressed per peroxisomal protein, no further calculations were needed. We assume a peroxisomal volume of approximately 1.8·10-6 L · mg of protein-1 (equal to that of mitochondria58). The structure of the active site of ACOX1 is essentially the same as that of MCAD32. This leads to a very similar binding of the substrate in both enzyme and a similar oxidation mechanism and FAD reduction. Because of this, and the lack of information on Km values for the products of ACOX1 we have used the of MCAD for the different enoyl-CoAs15. As mentioned in the results section of this paper, catalase only follows MM kinetics if the H2O2 concentration remains below a certain level, due to product inhibition. Given the high concentrations of H2O2 needed to reach this inhibition, we assume that the enzyme does follow MM kinetics. Moreover, because of its high Keq, the reaction was modelled as irreversible. Given the disparity in values in Vmax, we selected different papers about the isolated enzyme and averaged all the Vmax values. Moreover, we also took the value measured in human liver tissue extract and compared. Equilibrium constant values (Keq) were obtained, unless otherwise specified, from eQuilibrator (pH 7.5, pMg 3.0 and Ionic strength 0.25M).
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