Purification and Characterization of 3-Methylcrotonyl-CoA Carboxylase from Somatic Embryos of Daucus carota

3-Methylcrotonyl-CoA carboxylase, a biotin enzyme, was purified from embryos of Daucus carota. Polyethylene glycol precipitation and monomeric avidin affinity chromatography were used to purify all biotin enzymes from cell-free extracts of embryos. The resulting 3-methylcrotonyl-CoA carboxylase preparation had a specific activity of 745 nmol/min · mg protein, representing a 3725-fold purification of the enzyme and a 135% recovery of activity. Fractionation of the purified biotin-containing proteins by anionic exchange chromatography using Q-Sepharose partially resolved the 3-methylcrotonyl-CoA carboxylase from the other biotin enzymes. 3-Methylcrotonyl-CoA carboxylase has a biotin-contaming subunit with a molecular mass of about 78,000 Da and a non-biotin-containing subunit of about 65,000 Da. The native enzyme is 987,000 Da. The optimum pH for activity is between 8.0 and 8.4. The apparent Km values for the substrates 3-methylcrotonyl-CoA, sodium bicarbonate, and ATP are 42 ± 2 μM, 4.0 ± 0.9 mM, and 21 ± 2 μM, respectively. The enzyme is inhibited by acetoacetyl-CoA and palmitoyl-CoA.