Abstract
To study the functional cooperativity due to allosteric regulation in E.coli aspartate transcarbamoylase, two mutations (Lys60Glu & Lys94Glu) were introduced in the regulatory chain. This was done in order to disable the domain from binding the nucleotide effectors and at the same time would bring sufficient charge change on the surface of the protein molecule to be separated by ion-exchange chromatography. The mutant dimers along with the wild type dimers were used to reconstitute the holoenzyme hybrids. Due to different combinations four different types of hybrids were formed apparently exhibiting similar binding behavior to ion-exchange column. To purify these hybrid enzymes to homogeneity, preparative electrophoresis was successfully employed. The enzymes thus purified were assayed for comparison.
