β‐Methylcrotonyl‐CoA‐Carboxylase

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Abstract
β‐Methylcrotonyl‐CoA‐carboxylase was purified 120‐fold from an Achromobacter species. When the cells were grown on isovaleric acid, the enzyme was formed mainly during exponential growth. The enzyme can be labeled by addition of radioactive biotin to growing cells. A former procedure of purification was improved by using adsorption to calcium phosphate gel and chromatography on Sephadex G‐200. Enzyme homogeneous by both sedimentation and Tiselius electrophoresis was thus obtained in a yield of 40–70% and was crystallized in a form resembling crystalline propionyl‐CoA‐carboxylase. The molecular weight of the enzyme as determined by sedimentation velocity is 760000 daltons. The biotin content of 1,27 μmoles/g indicates 4 moles of biotin are bound to 1 mole of protein as is also the case for propionyl‐CoA‐carboxylase and pyruvate carboxylase. Glutamic and aspartic acid comprise 23% of the amino acids in the enzyme. Extrapolation of electrophoretic mobility as a function of pH indicates the isoelectric point of the enzyme is in the vicinity of pH 3.5. Such a value would mean that most of the carboxyl groups of the acidic residues are free. The usual methods of preparations for electron microscopy, even with preceding fixation steps, cause degradation of the enzyme since only particles of about 1/4 of the expected size could be seen. However, ultracentrifugation did not indicate any dissociation of the enzyme into subunits.