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

From the ¹ From the Department of Biological Chemistry, The University of Michigan, Ann Arbor, Michigan

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 + PP_i (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 PP_i concentrations are employed. The K_m of PP_i is 3.3 x 10^-2 m, and the enzyme is not saturated by PP_i concentrations approaching maximum solubility in the reaction mixture. The K_m of biotin for the exchange reaction (3.6 x 10^-9 m) is similar to that already known for holocarboxylase synthesis, whereas the K_m 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 PP_i, since the K_m of ATP for holocarboxylase synthesis is 2.1 x 10^-5 m when PP_i 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 PP_i (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 ß-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.

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