METHYLCROTONYL-COA CARBOXYLASE FROM BARLEY (HORDEUM-VULGARE L.) - PURIFICATION, KINETIC CHARACTERIZATION AND SENESCENCE-RELATED INDUCTION OF ACTIVITY

S-Methylcrotonyl-CoA carboxylase (MCC, 3-methylcrotonyl-CoA: carbondio xide ligase, (ADP-forming), EC 6.4.1.4) from barley shoots (Hordeum vu lgare L.) was purified to homogeneity with a 5340-foId purification fa ctor. MCC from barley consists of two subunits with a molecular weight of 60 and 85 kDa, and thus is a type II biotin-containing carboxylase . The larger polypeptide contains the biotin. Barley MCC has a native molecular weight of about 660 kDa. It exhibits maximum activity at 45 degrees C and at a pH of 8.0-8.3. Carboxylation of methylcrotonyl-CoA by barley MCC is Mg2+-dependent. Exchange of Mg2+ by Mn2+ or Fe2+ resu lted in only 13% or 20% of the original activity. Barley MCC was inhib ited to 95% by 1 mM acetoacetyl-CoA and to 25% by 1 mM palmitoyl-CoA. K-m-values for methylcrotonyl-CoA, ATP, and hydrogencarbonate were 31 mu M, 21 mu M and 2.8 mM, respectively. Kinetic measurements revealed an independent binding of methylcrotonyl-CoA and ATP to the enzyme, as well as sequential binding of methylcrotonyl-CoA vs. hydrogencarbonat e, and hydrogencarbonate vs. ATP. MCC-activity was low in young leaf t issue, but strongly increased with increasing age of the plants. For b arley seedlings an age of 21 days was found to be the optimum for enzy me isolation. In young barley shoots MCC-activity could be stimulated by treating the plants with leucine or senescence-inducing substances such as jasmonic acid. Apparent MCC-activity was also stimulated by co ntinuous darkness.