The Isolated Large Subunit of E. coli Carbamoylphosphate Synthetase Deviates from Kinetic Mechanism and Allosteric Behavior of the Holoenzyme

Abstract

Carbamoyl Phosphate Synthetase (CPS) from E. coli is a heterodimeric enzyme translated from the CarA and CarB genes. The polypeptides formed from these transcripts are 42kDa and 118kDa, respectively. CPS catalyzes the synthesis of carbamoylphosphate through activation of bicarbonate by one equivalent of MgATP priming bicarbonate for the addition of ammonia; the carbamate is then phosphorylated by a second equivalent of MgATP. The product of the CarB gene contains two active sites, both are functional as partial reactions, one is responsible for bicarbonate activation by MgATP and the amination of the activated bicarbonate using an ammonia source (glutamine in vivo), the second site is responsible for phosphorylation of carbamate and is distal to the heterodimer interface. The binding site for allosteric regulators is located on the large subunit proximal to the phosphorylation active site. The smaller subunit contains the active site for deamination of glutamine and is not mandatory for the activity of the sites on the large subunit. Previous work demonstrated the reverse of the phosphorylation partial reaction of CPS, formation of ATP from MgADP and carbamoylphosphate, is equilibrium ordered and responsive to allosteric ligands. In this work we provide evidence that removing the small subunit, thus relieving interfacial constraints 45A from the active site, results in reduced allosteric effects. Data shows that the kcat for the ATP synthesis reaction has decreased by 7-fold with no discernable difference in Kia. kcat = 6.9 sec-1 in holoenzyme and 1 sec-1 for the phosphorylation in isolated large subunit. The Kia determined for MgADP in holoenzyme is 510 µM and 700 µM in the isolated large subunit. Supported by the grant GM 33216 from the NIH.