Further Studies on the Properties of Liver Propionyl Coenzyme A Holocarboxylase Synthetase and the Specificity of Holocarboxylase Formation

Abstract

Abstract Several lines of evidence reported previously indicated that the reactions shown below are catalyzed by a synthetase purified from rabbit liver, but the predicted biotin-dependent 32P-labeled pyrophosphate-adenosine triphosphate exchange was found to be very slow. ATP + d-biotin (Mg++)/⇌ d-biotinyl 5'-adenylate + PPi (1) d-Biotinyl 5'-adenylate + propionyl coenzyme A apocarboxylase → propionyl-CoA holocarboxylase + AMP (2) In the present study it was found that a relatively rapid exchange reaction, dependent on the presence of Mg++ ions and the synthetase as well as biotin, occurs only if unusually high PPi concentrations are employed. The Km of PPi is 3.3 x 10-2 m, and the enzyme is not saturated by PPi concentrations approaching maximum solubility in the reaction mixture. The Km of biotin for the exchange reaction (3.6 x 10-9 m) is similar to that already known for holocarboxylase synthesis, whereas the Km of ATP for the exchange reaction (3.3 x 10-5 m) is about 50-fold greater than for holocarboxylase synthesis. This difference may be attributed to an inhibitory effect of PPi, since the Km of ATP for holocarboxylase synthesis is 2.1 x 10-5 m when PPi is included in the reaction mixture. The exchange rate under optimal conditions is slightly greater than the rate of ATP formation from d-biotinyl 5'-adenylate and PPi (reverse of Reaction 1) and is about 20 times greater than the rate of the over-all forward reaction in which biotin is covalently bound to the protein. The turnover number of the newly formed propionyl-CoA holocarboxylase is about 1280 moles of CO2 fixed per min per mole of biotin incorporated. The specificity of rabbit liver propionyl-CoA holocarboxylase synthetase, yeast acetyl-CoA holocarboxylase synthetase, and Propionibacterium shermanii holotranscarboxylase synthetase toward several apoenzymes (rat liver propionyl-CoA apocarboxylase, Commamonas terrigena β-methylcrotonyl-CoA apocarboxylase, and P. shermanii apotranscarboxylase) was determined. Significant activity was found with all synthetase-apocarboxylase combinations with the sole exception of the combination, liver synthetase-bacterial apotranscarboxylase. These results indicate broad synthetase specificity and suggest that the different apocarboxylases from diverse sources may have common structural features in the region of those lysine residues which accept biotin.