title: Synthesis of pyruvate carboxylase from its apoenzyme and (+)-biotin in Bacillus stearothermophilus. Purification and properties of the apoenzyme and the holoenzyme synthetase.
abstract: 1. Methods are described for the assay and purification of pyruvate apocarboxylase and pyruvate holocarboxylase synthetase from biotin-deficient Bacillus stearothermophilus. 2. Pyruvate apocarboxylase was obtained 200-fold purified and in a nearly homogeneous state; it closely resembled the holoenzyme of the thermophile in fractionation properties, electrophoretic mobility and molecular weight (estimated to be 350000 by gel filtration). 3. Pyruvate holocarboxylase synthetase, purified more than 50-fold, was estimated to have a molecular weight of approx. 40000. 4. The conversion of the purified apoenzyme into the holoenzyme required the presence of the synthetase, ATP (K(m)3.3x10(-7)m), (+)-biotin (K(m)7.5x10(-8)m) and Mg(2+); it differed from the conversions effected by systems forming other carboxylases in mesophilic organisms in also requiring the presence of acetyl-CoA.
mesh_terms: D000179:Acrylates; D000255:Adenosine Triphosphate / Q000378:metabolism; D001407:Bacillus / Q000201:enzymology*; D001710:Biotin / Q000378:metabolism*; D002845:Chromatography; D002850:Chromatography, Gel; D003065:Coenzyme A / Q000378:metabolism; D004589:Electrophoresis, Disc; D004792:Enzyme Precursors / Q000378:metabolism*; D005782:Gels; D008025:Ligases / Q000096:biosynthesis*; D008274:Magnesium; D008970:Molecular Weight; D011773:Pyruvates; D014461:Ultracentrifugation
chemical_list: D000179:Acrylates; D004792:Enzyme Precursors; D005782:Gels; D011773:Pyruvates; D001710:Biotin; D000255:Adenosine Triphosphate; D008025:Ligases; D008274:Magnesium; D003065:Coenzyme A