Random circular permutation leading to chain disruption within and near alpha helices in the catalytic chains of aspartate transcarbamoylase: effects on assembly, stability, and function.

摘要: A collection of circularly permuted catalytic chains aspartate transcarbamoylase (ATCase) has been generated by random circular permutation the pyrB gene. From library ATCases containing polypeptide chains, we have chosen for further investigation nine ATCase variants whose termini located within or close to an α helix. All fold and assemble into dodecameric holoenzymes with similar sedimentation coefficients slightly reduced thermal stabilities. Those disrupted three different helical regions in wild-type structure show no detectable enzyme activity apparent binding bisubstrate analog N-phosphonacetyl-l-aspartate. In contrast, two are just adjacent other helices catalytically active allosteric. As expected, disruptions more destabilizing than loop disruptions. Nonetheless, some lacking continuity can stable comprising six regulatory chains. For seven variants, is important but not necessary proper folding, assembly, stability holoenzyme.