The Purification and Properties of Dethiobiotin Synthetase

Abstract

Abstract The levels of dethiobiotin synthetase in Escherichia coli have been increased 6-fold over the wild type when prepared from a λ-lysogen carrying the bioD gene under phage control. The purification procedure employed gave a nearly homogeneous preparation, over 90% pure, with a 190-fold increase in specific activity. The enzyme has a molecular weight of about 42,000 and exists as a dimer. The stoichiometry of the reaction indicates that 1 mole each of ATP and CO2 are required and that ADP is the end product of the reaction. The Km values for each of the substrates were determined and ADP was found to be a competitive inhibitor. Only CTP can partially replace ATP while diaminobiotin is only 37% as effective as 7,8-diaminopelargonic acid. CO2 is the substrate in this reaction rather than bicarbonate (HCO3-). Carbamyl phosphate which gives some stimulation in cell-free extracts is inactive with the purified enzyme.