Purification and characterization of an alpha-ketoisocaproate oxygenase of rat liver

J Biol Chem. 1982 Jul 10;257(13):7460-7.

Abstract

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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Ascorbic Acid / pharmacology
  • Cations
  • Dioxygenases*
  • Dithiothreitol / pharmacology
  • Enzyme Activation
  • Hydrogen-Ion Concentration
  • Iron / pharmacology
  • Kinetics
  • Liver / enzymology*
  • Molecular Weight
  • Oxygenases / isolation & purification*
  • Rats

Substances

  • Cations
  • Iron
  • Oxygenases
  • Dioxygenases
  • alpha-ketoisocaproate oxygenase
  • Ascorbic Acid
  • Dithiothreitol