Nisha Sahay

Access Type

Open Access Dissertation

Date of Award


Degree Type


Degree Name



Biochemistry and Molecular Biology

First Advisor

Dr. David R. Evans


Pyrimidine nucleotides play a central role in cellular regulation. Most cells have two pathways to fulfill their pyrimidine nucleotide pools: the de novo and the salvage pathway. The de novo pyrimidine pathway begins with glutamine, ATP and bicarbonate, and through six-enzymatic steps yields uridine monophosphate (UMP). In mammals, the first three activities i.e. the glutamine-dependent carbamoyl phosphate synthetase (CPSase), aspartate transcarbamoylase (ATCase), and dihydroorotase (DHOase) are carried on a single 240-kDa polypeptide chain called CAD. Carbamoyl phosphate synthetase (CPSase), the first enzyme in the pathway catalyzes the synthesis of carbamoyl phosphate from two moles of ATP, bicarbonate, and ammonia derived from the hydrolysis of glutamine. The CPSase domain consists of two homologous halves, CPS.A and CPS.B, each of which is functionally equivalent, and as a homodimer can catalyze the overall CPSase reaction. Each of the two halves is further subdivided into subdomains A1, A2, A3, and B1, B2, and B3. CPSase is the locus of allosteric control in the mammalian de novo pathway. It undergoes feedback inhibition by UTP and activation by PRPP, a purine precursor that coordinates purine and pyrimidine biosynthesis, and by cAMP-dependent protein kinase phosphorylation. Several lines of evidence have demonstrated that the carboxyl end (B3) of the CPSase molecule is involved in allosteric regulation. My dissertation project involved the characterization of the B3 region. A chimera consisting of E. coli subdomains A1 and A2 fused to the mammalian B3 was constructed. Presence of the regulatory domain, B3, placed the CPS.A domain under allosteric control, demonstrating that the B3 domain behaves as an exchangeable ligand-binding module. The B3 domain was subsequently subcloned and expressed as a 26.7 kDa histidine-tagged recombinant. Binding studies with enzymatically synthesized, radiolabeled PRPP, demonstrated that the regulatory domain functioned as an autonomously folded, stable subdomain that bound PRPP. UTP binding was measured indirectly as an effect on PRPP binding. The regulatory domain formed a stoichiometric complex with the independently expressed CPS.A recombinant. The resulting hybrid was catalytically active and responded to allosteric effectors demonstrating that signals generated by the binding of effectors to B3 were transmitted to the CPS.A active site.