Synthesis of pyruvate carboxylase from its apoenzyme and (+)-biotin in Bacillus stearothermophilus. Mechanism and control of the reaction.
1. Acetyl-CoA acts as a positive allosteric effector in the formation of active pyruvate carboxylase from its apoenzyme, ATP and (+)-biotin which is catalysed by holoenzyme synthetase; this effect is counteracted by l-aspartate. 2. The Hill coefficients (apparent n values) were approximately 2 for acetyl-CoA and 4 for l-aspartate; the n value for each effector remained constant when the concentration of the other effector was varied. 3. Active pyruvate carboxylase was formed also when the apoenzyme was incubated with holoenzyme synthetase and synthetic biotinyl-5'-AMP; acetyl-CoA and l-aspartate affected this process as they did the overall reaction from (+)-biotin and ATP. 4. When hydroxylamine replaced the apoenzyme, holoenzyme synthetase catalysed the formation of biotinylhydroxamate from (+)-biotin and ATP. This reaction was not affected by the allosteric effectors. 5. The apoenzyme was protected against thermal denaturation by acetyl-CoA and, to a lesser degree, by l-aspartate. The holoenzyme synthetase was not markedly protected by these effectors. 6. It is concluded that the allosteric effectors act on the apoenzyme and not the synthetase.
D000255:Adenosine Triphosphate; D001224:Aspartic Acid; D001407:Bacillus / Q000378:metabolism*; D001710:Biotin / Q000378:metabolism*; D002247:Carbon Isotopes; D002845:Chromatography; D003065:Coenzyme A; D004792:Enzyme Precursors / Q000378:metabolism*; D006898:Hydroxylamines; D008025:Ligases / Q000096:biosynthesis*; D011773:Pyruvates; D013053:Spectrophotometry; D013696:Temperature
D002247:Carbon Isotopes; D004792:Enzyme Precursors; D006898:Hydroxylamines; D011773:Pyruvates; D001224:Aspartic Acid; D001710:Biotin; D000255:Adenosine Triphosphate; D008025:Ligases; D003065:Coenzyme A