Publications

• Expression, purification, characterization of human 3-methylcrotonyl-CoA carboxylase (MCCC).

  Chu C.H., Cheng D.

  The current study reports the use of baculovirus system to express functionally active human recombinant 3-methylcrotonyl-CoA carboxylase (MCCC), a heteromultimeric complex that is composed of alpha and beta subunits which are encoded by distinct genes. Using immuno-affinity purification, an effic ... >> More

  The current study reports the use of baculovirus system to express functionally active human recombinant 3-methylcrotonyl-CoA carboxylase (MCCC), a heteromultimeric complex that is composed of alpha and beta subunits which are encoded by distinct genes. Using immuno-affinity purification, an efficient protocol has been developed to purify the active MCCC which appears to reside in a approximately 500-800kDa complex in Superpose-6 gel-filtration chromatography. Consistent with the native enzyme, in the recombinant human MCCC, the stoichiometry of alpha and beta subunits are at a one:one ratio. The k(cat) value of the recombinant enzyme is determined to be approximately 4.0s(-1). It also possesses K(m) values (ATP: 45+/-11microM; 3-methylcrotonyl-CoA: 74+/-7microM) similar to those reported for the native enzyme. The recombinant human MCCC described here may provide a counter-screen enzyme source for testing cross reactivity for inhibitors against acetyl-CoA carboxylases which are designed to treat obesity, type 2 diabetes and other metabolic disorders. << Less

  Protein Expr. Purif. 53:421-427(2007) [PubMed] [EuropePMC]

• Purification and characterization of 3-methylcrotonyl-coenzyme-A carboxylase from leaves of Zea mays.

  Diez T.A., Wurtele E.S., Nikolau B.J.

  3-Methylcrotonyl-CoA carboxylase has been purified to near homogeneity from maize leaves. The resulting preparations of 3-methylcrotonyl-CoA carboxylase have a specific activity of between 200 and 600 nmol.min-1.mg-1 protein, representing an approximately 5000-fold purification of the enzyme. The ... >> More

  3-Methylcrotonyl-CoA carboxylase has been purified to near homogeneity from maize leaves. The resulting preparations of 3-methylcrotonyl-CoA carboxylase have a specific activity of between 200 and 600 nmol.min-1.mg-1 protein, representing an approximately 5000-fold purification of the enzyme. The purified 3-methylcrotonyl-CoA carboxylase has a molecular weight of 853,000 +/-34,000 and is composed of two types of subunits, a biotin-containing subunit of 80 +/- 2 kDa and a non-biotin-containing subunit of 58.5 +/-1.5 kDa. These data suggest that the enzyme has an alpha 6 beta 6 configuration. The optimum pH for activity is 8.0. The kinetic constants for the substrates 3-methylcrotonyl-CoA, ATP, and HCO3-are 11 microM, 20 microM, and 0.8 mM, respectively. Kinetic studies of the 3-methylcrotonyl-CoA carboxylase reaction with variable concentrations of two substrates confirmed that ATP and HCO3-bind sequentially to the enzyme and that ATP and 3-methylcrotonyl-CoA bind in ping-pong fashion. However, similar analyses indicate that the binding of HCO3-at the first site is affected by 3-methylcrotonyl-CoA. Kinetic studies of the role of Mg2+ in the 3-methylcrotonyl-CoA carboxylase reaction establish that Mg.ATP is the substrate for the enzyme, that free ATP is an inhibitor, and that free Mg2+ is an activator. Both Mn2+ and Co2+ can substitute somewhat for Mg2+, but Zn2+ is unable to do so. In addition to carboxylating 3-methylcrotonyl-CoA, the maize carboxylase can carboxylate crotonyl-CoA, but not acetoacetyl-CoA. In fact, acetoacetyl-CoA is a potent, noncompetitive inhibitor, which indicates that the enzyme contains an acetoacetyl-CoA binding site that is independent of the active sites. The monovalent cations K+, Cs+, Rb+, and NH4+ activated 3-methylcrotonyl-CoA carboxylase activity, with Rb+ being the most potent activator. The inhibition of 3-methylcrotonyl-CoA carboxylase by sulfhydryl and arginyl modifying reagents could be partly alleviated by the substrates ATP and 3-methylcrotonyl-CoA, which suggests that sulfhydryl and arginyl residues may be involved in catalysis. << Less

  Arch Biochem Biophys 310:64-75(1994) [PubMed] [EuropePMC]

• Purification and Characterization of 3-Methylcrotonyl-Coenzyme A Carboxylase from Higher Plant Mitochondria.

  Alban C., Baldet P., Axiotis S., Douce R.

  3-Methylcrotonyl-coenzyme A (CoA) carboxylase was purified to homogeneity from pea (Pisum sativum L.) leaf and potato (Solanum tuberosum L.) tuber mitochondria. The native enzyme has an apparent molecular weight of 530,000 in pea leaf and 500,000 in potato tuber as measured by gel filtration. Poly ... >> More

  3-Methylcrotonyl-coenzyme A (CoA) carboxylase was purified to homogeneity from pea (Pisum sativum L.) leaf and potato (Solanum tuberosum L.) tuber mitochondria. The native enzyme has an apparent molecular weight of 530,000 in pea leaf and 500,000 in potato tuber as measured by gel filtration. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate disclosed two nonidentical subunits. The larger subunit (B subunit) is biotinylated and has an apparent molecular weight of 76,000 in pea leaf and 74,000 in potato tuber. The smaller subunit (A subunit) is biotin free and has an apparent molecular weight of 54,000 in pea leaf and 53,000 in potato tuber. The biotin content of the enzyme is 1 mol/133,000 g of protein and 1 mol/128,000 g of protein in pea leaf and potato tuber, respectively. These values are consistent with an A4B4 tetrameric structure for the native enzyme. Maximal 3-methylcrotonyl-CoA carboxylase activity was found at pH 8 to 8.3 and at 35 to 38[deg]C in the presence of Mg2+. Kinetic constants (apparent Km values) for the enzyme substrates 3-methylcrotonyl-CoA, ATP, and HCO3-were: 0.1 mM, 0.1 mM, and 0.9 mM, respectively, for pea leaf 3-methylcrotonyl-CoA carboxylase and 0.1 mM, 0.07 mM, and 0.34 mM, respectively, for potato tuber 3-methylcrotonyl-CoA carboxylase. A steady-state kinetic analysis of the carboxylase-catalyzed carboxylation of 3-methylcrotonyl-CoA gave rise to parallel line patterns in double reciprocal plots of initial velocity with the substrate pairs 3-methylcrotonyl-CoA plus ATP and 3-methylcrotonyl-CoA plus HCO3- and an intersecting line pattern with the substrate pair HCO3-plus ATP. It was concluded that the kinetic mechanism involves a double displacement. Purified 3-methylcrotonyl-CoA carboxylase was inhibited by end products of the reaction catalyzed, namely ADP and orthophosphate, and by 3-hydroxy-3-methylglutaryl-CoA. Finally, as for the 3-methylcrotonyl-CoA carboxylases from mammalian and bacterial sources, plant 3-methylcrotonyl-CoA carboxylase was sensitive to sulfhydryl and arginyl reagents. << Less

  Plant Physiol 102:957-965(1993) [PubMed] [EuropePMC]