Acetyl‐Coenzyme‐A Carboxylase of Candida lipolytica

Abstract

Acetyl-coenzyme-A carboxylase [EC 6.4.1.2] was isolated in homogeneous form from C. lipolytica. The homogeneity of the enzyme preparation is evidence by analytical ultracentrifugation, dodecylsulfate-polyacrylamide gel electrophoresis and Ouchterlony double-diffusion analysis. The purified enzyme exhibits a specific activity of 8.0 U[units]/mg protein at 25.degree. C and contains 1 mol biotin/263,000 g protein. The sedimentation coefficient (s20,w) of the enzyme is 18 S. It was shown by dodecylsulfate-polyacrylamide gel electrophoresis that the enzyme possesses only 1 kind of subunit with a MW of 230,000. Apparently the C. lipolytica enzyme has a highly integrated subunit structure. The C. lipolytica enzyme is labile, but is stabilized by glycerol. The enzyme is markedly activated by polyethylene glycol, the activation being due principally to a decrease in the Km values for substrates. Even in the presence of this activator, the Km value for acetyl-CoA of the C. lipolytica enzyme is higher than that of the enzyme from Saccharomyces cerevisiae and animal tissues. The C. lipolytica enzyme, unlike the enzyme from animal tissues, is not activated by citrate.