136 Chapter 5 reported very different results depending on the methods and assays employed. While catalase has been reported not to follow Michaelis Menten kinetics throughout the entire range of substrates 38,39, it has also been reported to do so when the concentration of H2O2 is kept below 200 mM. At higher concentrations, hydrogen peroxide inactivates the enzyme, which has been suggested to alter apparent Km and Vmax values. Most kinetic assays of catalase performed in the early days, where done in the presence of supraphysiological concentrations H2O2 (up to molar range), which might have affected the results40. Apparent K m values vary between 40 to almost 600 mM39, while the apparent kcat ranges between 55,000 to over 800,000 s-1 39. Moreover, the K eq and the second order rate of the reaction have also been reported to be very high41. While high variability between values is observed, all the measured V max and Km are extremely high, reflecting the high turnover of the enzyme and the fact that hydrogen peroxide is shortly-lived in the peroxisome 42. Bifunctional Protein The second and third reactions of the peroxisomal β-oxidation are catalyzed by a bifunctional enzyme, which performs first a hydration reaction converting the 2-enoyl-CoA into a 3-hydroxyacyl-CoA, and then a dehydrogenation of which the final product is a 3-ketoacyl-CoA. There are two isoenzymes, usually referred to as L- and D- bifunctional protein (LBP and DBP), but sometimes as peroxisomal multifunctional enzyme type I and II (MFE-I and MFE-II)43,44. 39 Most peroxisomal substrates are metabolized by the D-bifunctional protein (DBP), while the L-form metabolizes preferentially dicarboxylic acids45. Specifically, the D-functional protein metabolizes very-long-chain enoylCoAs, branched chain fatty acids and bile acid intermediates44,46 . The enzyme donates the electrons to NAD+ in order to produce NADH. Jiang et al44,47, purified both DBP and LBP from human liver and studied their characteristics. They showed that while both enzymes were present in similar amounts and had similar specific activities for their respective substrates, the D-form had a higher activity towards longer carbon-chain substrates than the L-form. DBP showed its maximal activity towards decanoyl-CoA and dodecanoyl-CoA. Peroxisomal 3-ketoacyl-CoA thiolase 1 (ACAA1) The fourth and last step of the peroxisomal β-oxidation is catalyzed by different thiolase isoenzymes. These enzymes catalyze the thiolytic cleavage of 3-ketoacyl-CoAs, which yields an acyl-CoA that is shortened by two carbons plus acetyl-CoA. Mouse and rat peroxisomes contain three different 3-ketoacylCoA thiolases, namely thiolase A, thiolase B, and SCP-2/3-ketoacyl-CoA thiolase
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