Purification and characterization of an alpha-ketoisocaproate oxygenase of rat liver.
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.
D000818:Animals; D001205:Ascorbic Acid / Q000494:pharmacology; D002412:Cations; D049308:Dioxygenases; D004229:Dithiothreitol / Q000494:pharmacology; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000494:pharmacology; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008970:Molecular Weight; D010105:Oxygenases / Q000302:isolation & purification*; D051381:Rats
D002412:Cations; D007501:Iron; D010105:Oxygenases; D049308:Dioxygenases; C034776:alpha-ketoisocaproate oxygenase; D001205:Ascorbic Acid; D004229:Dithiothreitol