ALPHA-OXIDATION OF 2-HYDROXYSTEARIC ACID IN VITRO

The properties of the solubilized rat brain microsomal enzyme that oxidatively decarboxylates 2-hydroxystearic acid are described. Fe2+ is required in addition to O2 and enzymatic activity is optimum at pH 6.1 in phosphate buffer or at pH 6.9 in Tris buffer, is stimulated by peroxide-generating systems and is inhibited by catalase but not by sodium azide. Ascorbate enhances enzymatic activity only when metal ion is present but does not serve as a cofactor in the absence of metal ion. In addition, non-enzymatic decarboxylation reactions are stimulated by ascorbate in the presence of metal ion and by metal ions alone. Adenine and pyridine nucleotides stimulate the decarboxylation reaction when microsomes, but not when microsomal sonicates, are used as the enzyme source. Similarly, FAD and CoA do not serve as cofactors. EDTA, o-phenanthroline, and 2,2'-dipyridyl inhibit enzyme activity, lending support to the hypothesis that Fe2+ is the required metal ion for enzymatic activity although other metal ions also catalyzed an enzymatic decarbocylation reaction. Sulfhydryl groups may be present at the active site of the enzyme since p-chloromercuribenzoate inhibited the release of C02. A radioactive peak corresponding to 2-ketostearic acid was detected by thin-layer and gas chromatographic analysis when microsomes plus supernatant fraction were incubated with substrate. The following reaction sequence is suggested: RCHOHCOOH+enzyme+O2 → Fe2+, [RCOCOOH] enzyme-H2O2 → Fe2+RCOOH+CO2+H2O. © 1968.