Perturbations to the Allosteric Regulation of Carbamoyl Phosphate Synthetase upon Removal of Interfacial Restraints

Abstract

Carbamoyl phosphate synthetase (CPS) from E. coli is an ?? heterodimeric allosterically regulated enzyme serving as a gatekeeper for entry into both the de novo pyrimidine biosynthesis pathway and the urea cycle (in ureotelic organisms). The regulation of CPS by the activator L-ornithine and inhibitor UMP is an entropy dominated phenomenon. Liberation of the heterodimeric interface by removal of the glutaminase subunit creates increased conformational freedom at the interface and maintains the functionality of the two reaction centers located in the large subunit. The liberation of the interface has profound effects on the allosteric regulation of the two reaction centers and causes a perturbation in the mechanism of the ATP synthesis reaction when investigated with steady state kinetics.

Van’t Hoff analysis of ??_????, the coupling parameter, demonstrated ornithine binding generates a decrease in ???_????, the coupling free energy, of the bicarbonate dependent ATPase reaction of 1.1 kcal/mol in the absence of the glutaminase subunit indicating an augmentation of ornithine’s ability to activate CPS from E. coli. The loss of the glutaminase subunit results in the ???_???? of activation of the ATP synthesis reaction being increased by 0.8 kcal/mol resulting in attenuation of activation. Quantification of ??_???? and determination of ???_???? of inhibition by UMP demonstrated inhibition of MgATP binding by UMP is also augmented in the absence of the glutaminase subunit, with an increase of 0.44 kcal/mol while data suggests the inhibition of the ATP synthesis reaction is abolished upon removal of the glutaminase subunit.

Consequently, releasing the structural restraints imposed by a subunit interaction with no catalytic constraints can have substantial, though unpredictable effects on llosteric communication in E. coli CPS.