134 Chapter 5 Acyl-CoA oxidase 1 Acyl-CoA oxidase 1, the first enzyme of the peroxisomal β-oxidation pathway is commonly referred to as the rate limiting step26,27. This statement is based on studies reporting that the Km values of the enzyme for certain substrates such as lauroyl-, myristoyl-, palmitoyl- and stearoyl-CoA27 are almost the same as the ones observed for the whole pathway28. Moreover, the variations in V max reported by Hovik28 in the whole pathway were described to be very similar to those reported for ACOX127. These results, together with a similar pattern in substrate inhibition, have been used to define ACOX1 as the rate-limiting step of the pathway. Formal proof of this statement would require Metabolic Control Analysis29. Rat liver peroxisomes have at least three acyl-CoA oxidases. ACOX1 shows activity towards straight-chain acyl-CoAs, including mono- and dicarboxylic fatty acids. ACOX2 metabolizes bile acid intermediates. Finally, ACOX3, traditionally known as pristanoyl-CoA oxidase, is involved in the oxidation of 2-methyl-branched-chain fatty acids30. Interestingly, human peroxisomes only have ACOX1 and a second branched-chain acyl-CoA oxidase, which oxidizes 2-methyl-branched-chain acyl-CoAs as well as straight-chain acyl-CoAs (dicarboxylic VLCFA). Given the complexity of the system, and the lack of kinetic parameters except for ACOX1, we focus on the oxidation of straight-chain acylCoAs performed by ACOX1 here 10. Acyl-CoA Oxidase 1 is synthesized as a polypeptide, which is proteolytically cleaved into the two smaller (functional) peptides upon import into the peroxisome27. The enzyme introduces a double bond between C2 and C3 of the acyl-CoA substrate, thus converting it into a 2-enoyl-CoA. ACOX1 is a flavoprotein that contains an FAD group. This FAD group is reduced into FADH2 upon the oxidation of an acyl-CoA into a 2-enoyl CoA. The electrons are then donated to molecular oxygen, which in turn is reduced to hydrogen peroxide. This reaction is different from that catalyzed by the first enzyme of mitochondrial β-oxidation (acyl-CoA dehydrogenase), which ultimately donates the electrons to the electron transport chain to produce ATP31. Crystallography studies of ACOX1 demonstrated that the structure of the enzyme is similar to that of the medium-chain acyl-CoA dehydrogenase (MCAD)32, particularly, at its active site. Although both enzymes contain an FAD molecule, the flavin ring is very protected in MCAD, which makes it unreachable to molecular oxygen. In the case of ACOX1 this ring is in a large cavity, where it is accessible to molecular oxygen. Interestingly, the binding site between MCAD and the electron transport flavoprotein, which donates the electrons
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