Synthesis of pyruvate carboxylase from its apoenzyme and (+)-biotin in Bacillus stearothermophilus. Purification and properties of the apoenzyme and the holoenzyme synthetase.
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.
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
D000179:Acrylates; D004792:Enzyme Precursors; D005782:Gels; D011773:Pyruvates; D001710:Biotin; D000255:Adenosine Triphosphate; D008025:Ligases; D008274:Magnesium; D003065:Coenzyme A