Sabourin P J, Bieber L L
J Biol Chem. 1982 Jul 10;257(13):7460-7.
Rat liver contains a cytosolic alpha-ketoisocaproate oxygenase which oxidatively decarboxylates and hydroxylates alpha-ketoisocaproate to form beta-hydroxyisovalerate. This oxygenase was purified to near homogeneity. The oxygenase is unstable during purification, unless 5% monothioglycerol is added. The purified enzyme is stable in the presence of 5% monothioglycerol for 3 weeks at 4 degrees C and at least 10 weeks at -80 degrees C. The molecular weight of the alpha-ketoisocaproate oxygenase as determined to be 46,000 and 51,000 using denaturing and nondenaturing conditions, respectively, indicating a monomer. The alpha-ketoisocaproate oxygenase requires Fe2+; other metal ions did not replace Fe2+. Ascorbate activates the enzyme at subsaturating levels of Fe2+, by regenerating Fe2+. The activity is markedly affected by the type of buffer used. For example, the oxygenase activity increased 2- to 3-fold when 0.1 M maleate was used. Iron chelators, such as ADP and EDTA, are inhibitory. The ratio of decarboxylation of 1 mM alpha-[1-14C] ketoisocaproate (as measured by 14CO2 release) to decarboxylation of 1 mM alpha-[1-14C]keto-gamma-methiolbutyrate is 1.0 for all purification fractions, indicating that a single enzyme catalyzes the decarboxylation of both substrates. The apparent Km and Vmax values of the alpha-ketoisocaproate oxygenase using optimized assay conditions are 0.32 mM and 130 nmol/min/mg of protein for alpha-ketoisocaproate and 1.9 mM and 247 nmol/min/mg of protein for alpha-keto-gamma-methiolbutyrate. The principal product of the purified alpha-ketoisocaproate oxygenase, using alpha-ketoisocaproate as a substrate, is beta-hydroxyisovalerate, although small amounts of a compound, which has the chromatographic properties of isovalerate, are also produced.
大鼠肝脏含有一种胞质α-酮异己酸加氧酶，该酶可将α-酮异己酸氧化脱羧并羟基化，形成β-羟基异戊酸。这种加氧酶被纯化至接近均一状态。在纯化过程中，该加氧酶不稳定，除非添加5%的单硫甘油。纯化后的酶在5%单硫甘油存在下，于4℃可稳定保存3周，在-80℃至少可稳定保存10周。使用变性和非变性条件测定，α-酮异己酸加氧酶的分子量分别为46,000和51,000，表明其为单体。α-酮异己酸加氧酶需要Fe2+；其他金属离子不能替代Fe2+。抗坏血酸通过再生Fe2+，在Fe2+亚饱和水平下激活该酶。酶的活性受所用缓冲液类型的显著影响。例如，使用0.1 M马来酸时，加氧酶活性增加2至3倍。铁螯合剂，如ADP和EDTA，具有抑制作用。对于所有纯化级分，1 mMα-[1-14C]酮异己酸的脱羧作用（通过14CO2释放量测定）与1 mMα-[1-14C]酮-γ-甲硫基丁酸的脱羧作用之比为1.0，表明单一酶催化两种底物的脱羧反应。使用优化的测定条件，α-酮异己酸加氧酶对α-酮异己酸的表观Km和Vmax值分别为0.32 mM和130 nmol/min/mg蛋白质，对α-酮-γ-甲硫基丁酸的表观Km和Vmax值分别为1.9 mM和247 nmol/min/mg蛋白质。以α-酮异己酸为底物时，纯化后的α-酮异己酸加氧酶的主要产物是β-羟基异戊酸，不过也会产生少量具有异戊酸色谱特性的化合物。
