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-containing 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 microM, 4.0 +/- 0.9 mM, and 21 +/- 2 microM, respectively. The enzyme is inhibited by acetoacetyl-CoA and palmitoyl-CoA.
Department of Food Science, Iowa State University, Ames, Iowa 50011.
Y
Y
Chen
E S
ES
Wurtele
X
X
Wang
B J
BJ
Nikolau
D000214:Acyl Coenzyme A / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001639:Bicarbonates / Q000378:metabolism / Q000494:pharmacology; D001710:Biotin / Q000032:analysis; D019735:Carbon-Carbon Ligases; D005591:Chemical Fractionation; D011232:Chemical Precipitation; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008025:Ligases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D011092:Polyethylene Glycols; D012639:Seeds / Q000201:enzymology*; D012964:Sodium / Q000378:metabolism / Q000494:pharmacology; D017693:Sodium Bicarbonate; D013379:Substrate Specificity; D014675:Vegetables
D000214:Acyl Coenzyme A; D001639:Bicarbonates; D011092:Polyethylene Glycols; C033618:tiglyl-coenzyme A; D001710:Biotin; D000255:Adenosine Triphosphate; D017693:Sodium Bicarbonate; D012964:Sodium; D008025:Ligases; D019735:Carbon-Carbon Ligases; C021573:methylcrotonoyl-CoA carboxylase