PURIFICATION AND CHARACTERIZATION OF 3-METHYLCROTONYL-COENZYME-A CARBOXYLASE FROM LEAVES OF ZEA-MAYS

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 o f the enzyme. The purified 3-methylcrotonyl-CoA carboxylase has a mole cular weight of 853,000 +/- 34,000 and is composed of two types of sub units, a biotin-containing subunit of 80 +/- 2 kDa and a non-biotin-co ntaining subunit of 58.5 +/- 1.5 kDa. These data suggest that the enzy me has an alpha6beta6 configuration. The optimum pH for activity is 8. 0. The kinetic constants for the substrates 3-methylcrotonyl-CoA, ATP, and HCO3- are 11 muM, 20 muM, and 0.8 mM, respectively. Kinetic studi es of the 3-methylcrotonyl-CoA carboxylase reaction with variable conc entrations of two substrates confirmed that ATP and HCO3- bind sequent ially 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 s tudies of the role of Mg2+ in the 3-methylcrotonyl-CoA carboxylase rea ction establish that Mg . ATP is the substrate for the enzyme, that fr ee ATP is an inhibitor, and that free Mg2+ is an activator. Both Mn2and Co2+ can substitute somewhat for Mg2+, but Zn2+ is unable to do so . In addition to carboxylating 3-methylerotonyl-CoA, the maize carboxy lase can carboxylate crotonyl-CoA, but not acetoacetyl-CoA. In fact, a cetoacetyl-CoA is a potent, noncompetitive inhibitor, which indicates that the enzyme contains an acetoacetyl-CoA binding site that is indep endent of the active sites. The monovalent cations K+, Cs+, Rb+, and N H4+ activated 3-methylcrotonyl-CoA carboxylase activity, with Rb+ bein g the most potent activator. The inhibition of 3-methylcrotonyl-CoA ca rboxylase by sulfhydryl and arginyl modifying reagents could be partly alleviated by the substrates ATP and 3-methylcrotonyl-CoA, which sugg ests that sulfhydryl and arginyl residues may be involved in catalysis . (C) 1994 Academic Press, Inc.