Defense Date


Graduation Date

Fall 2012


Immediate Access

Submission Type


Degree Name



Chemistry and Biochemistry

Committee Chair

Jeffrey Evanseck

Committee Member

Jeffry Madura

Committee Member

Rita Mihailescu

Committee Member

Steven Firestine


Carboxyphosphate, Charge assisted hydrogen bond, ONIOM, Resonance assisted hydrogen bond


N5-CAIR synthetase catalyzes the conversion of 5-amino-imidazole ribonucleotide (AIR), Mg2ATP, and bicarbonate into N5-CAIR, Mg2ADP, and inorganic phosphate. DFT and ONIOM QM/MM computations revealed that the first step in the reaction mechanism of N5-CAIR synthetase involves the formation of dianionic carboxyphosphate (CP) from the attack of bicarbonate on the γ- phosphate of Mg2ATP, through an SN2 type transition structure. Properties of CP were never reported, as this compound is extremely unstable (t1/2 of 70 ms). Therefore, high level ab inito (MP2 and CCSD(T)), and DFT (B3LYP, BB1K, M05-2X, M06-2X and MPW1K) methods were used to investigate the structure and energetics of CP in vacuum, and PCM continuum solvation model. We report, for the first time, that carboxyphosphate adopts a "pseudo-cyclic" conformation with an intramolecular resonance assisted hydrogen bonded (RAHB) six membered ring and calculations reveal that this conformation is found to be the most stable in vacuum and solvent. M062X/aug-cc-pVTZ was observed to deliver structure and energetics that are in excellent agreement with high level ab initio methods. Characterization of the catalytic mechanism of N5-CAIR synthetase involves the decomposition of CP into carbon dioxide and inorganic phosphate. Our calculations on the decomposition of dianionic CP showed that the energetic pathway was unreasonable and unlikely in the active site. Therefore, conformations and energies of monoanionic CP were determined. Two stable pseudo-cyclic conformations and a pseudo-bicylic conformation were identified to be at least 12 kcal/mol stable than their 11 different linear counterparts. Potential energy surface calculations revealed that the decomposition occurs through intramolecular proton transfer from the acid to phosphate side, which triggers decarboxylation. ONIOM(M062X/6-31G(d):AMBER) calculations suggest that, dianionic CP in the active site is converted into the monoanionic form by accepting a proton from Lys353. Next, CP attains a specific pseudo-cyclic conformation stabilized by Lys353 to trigger decarboxylation. In the final step of carboxylation of AIR, DFT and ONIOM(DFT:AMBER) calculations predict that in a concerted manner the amine of AIR attacks the activated CO2 and Asp153 removes its proton. Computational results are in accord with site-directed mutagenesis studies.