Photoaffinity labeling and stoichiometry of the coenzyme A ester sites of transcarboxylase.

Abstract

SUMMARY AND DISCUSSION Binding Studies with Methylmalonyl-CoA - Our first ef- forts in determining the stoichiometry of the CoA ester sites of transcarboxylase were with propionyl-CoA and methylma- lonyl-CoA. We found that propionyl-CoA could not be used because both transcarboxylase and the 12 S,, subunit catalyze the deacylation of propionyl-CoA (Equation 4). The rate of hydrolysis was 18 to 30 nmol/min/mg at room temperature and 0.4 to 1 nmol/min/mg at 0 to 5”. This rate is approximately 1000 times slower than the rate of the transcar- boxylation but too fast, even at cold temperatures, for the binding experiments. Likewise, methylmalonyl-Cob could not be used with trans- carboxylase because the biotin of the enzyme is carboxylated by the methylmalonyl-CoA and there is spontaneous decar- boxylation of the enzyme. biotin . CO, complex (8) (Equation 5). co, Enz-biotin Enz-biotin-COO coo- COSCoA 1 ‘/ CH,CH,COSCoA (5) / ‘1 CH, H