132 Chapter 5 regulation of enzyme concentrations, and nutritional and pharmacological interventions13,14. While several kinetic models have been constructed and experimentally validated for mitochondrial metabolism15–17, no kinetic model of peroxisomal fatty-acid metabolism has been reported yet. One of the biggest challenges for the establishment of a peroxisomal β-oxidation model is the relatively limited availability of reliable kinetic parameters for the enzymes involved in the pathway. Most of the kinetic studies date back to the early 90s and lack information on oxidation of very-long-chain substrates due to experimental limitations caused by the extremely low solubility of these compounds18. Another limiting factor is the lack of kinetic information on peroxisomal transporters. The aim of this study is to review the kinetics that are available for the different enzymes and transporters involved in the peroxisomal β-oxidation and use this knowledge to build a first kinetic model of the pathway in liver cells. Subsequently, we present a case study in which we use the model to predict the effect of amino-acid restriction on the pathway flux and metabolite concentrations. To do so, proteomics data from in vitro studies were integrated into the model, thereby generating a context-specific model. RESULTS AND DISCUSSION Review of kinetics of enzymes involved in the peroxisomal β-oxidation We first present a detailed review of the kinetics of the different enzymes and transporters involved in the β-oxidation of straight-chain acyl-CoAs in the peroxisome. The activation of fatty acids by acyl-CoA synthetases19 is outside the scope of this work. We have evaluated different studies based on their relevance for in vivo conditions. The papers selected, and hereby discussed, were chosen based on their physiological relevance. Enzymatic assays measured at supraphysiological conditions (pH, temperature, H2O2 concentration, etc.) were not considered. In cases when one study did not provide sufficient kinetic data, two or more studies were combined. Since there is no complete dataset for any single organism, we included information obtained from mouse, rat and human liver, and when relevant also from other tissues or organisms. Once a saturated acyl-CoA is inside the peroxisome, it is oxidized by ACOX1 into a 2-trans-enoyl CoA. This reaction is coupled to the oxidation of molecular
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