Computational Characterization of Carboxyphosphate

Carboxyphosphate (CP) is an important intermediate involved in reactions catalyzed by acetyl-CoA carboxylase, pyruvate carboxylase, N5-Carboxyaminoimidazole ribonucleotide synthetase, propionyl-CoA carboxylase, urea amidolyase, and carbamoyl phosphate synthetase. Despite its important role, properties for CP have never been reported due to its short estimated half-life (t1/2 70 ms). Thus, the high level ab initio methods, MP2 and CCSD(T), along with the DFT functionals: B3LYP, BB1K, M05-2X, M06-2X, and MPW1K were used to investigate the structure and energetics of CP in both vacuum and the PCM continuum solvation model of water. It was found that CP adopts a novel pseudo-cyclic structure featuring an intramolecular charge-assisted hydrogen bond (CAHB) that is reminiscent of chair cyclohexane. This structure is found to be the most stable in both vacuum and implicit solvation for both mono and dianionic charge states. Additionally, the M06-2X/aug-cc-pVTZ level of theory was shown to give consistent agreement with ab initio methods for both geometric and energetic properties. The strengths of the CAHBs observed in mono- and dianionic CP were estimated to be within the range of -17.8 to -25.4 and -15.7 to -20.9 kcal/mol, respectively. This classifies them as short-strong but not low-barrier and makes them the dominant stabilizing feature for these conformations. pKa values were computed to distinguish between different possible protonation states of CP. The predicted pKa values were found to be be -3.43±0.81, 4.04±0.35, and 8.14±1.92 for the first, second and third acid dissociations of CP, respectively, indicating it is most likely to be present as a dianion or trianion in aqueous solution but more work is required to predict its charge state in the enzymatic pocket

Intramolecular Charge-Assisted Hydrogen Bond Strength in Pseudochair Carboxyphosphate

Carboxyphosphate, a suspected intermediate in ATP-dependent carboxylases, has not been isolated nor observed directly by experiment. Consequently, little is known concerning its structure, stability, and ionization state. Recently, carboxyphosphate as either a monoanion or dianion has been shown computationally to adopt a novel pseudochair conformation featuring an intramolecular charge-assisted hydrogen bond (CAHB). In this work, additive and subtractive correction schemes to the commonly employed open–closed method are used to estimate the strength of the CAHB. Truhlar’s Minnesota M06-2X functional with Dunning’s aug-cc-pVTZ basis set has been used for geometry optimization, energy evaluation, and frequency analysis. The CHARMM force field has been used to approximate the Pauli repulsive terms in the closed and open forms of carboxyphosphate. From our additive correction scheme, differential Pauli repulsion contributions between the pseudochair (closed) and open conformations of carboxyphosphate are found to be significant in determining the CAHB strength. The additive correction modifies the CAHB prediction (Δ<i>E</i>_{closed–open}) of −14 kcal/mol for the monoanion and −12 kcal/mol for the dianion to −22.9 and −18.4 kcal/mol, respectively. Results from the subtractive technique reinforce those from our additive procedure, where the predicted CAHB strength ranges from −17.8 to −25.4 kcal/mol for the monoanion and from −15.7 to −20.9 kcal/mol for the dianion. Ultimately, we find that the CAHB in carboxyphosphate meets the criteria for short-strong hydrogen bonds. However, carboxyphosphate has a unique energy profile that does not result in the symmetric double-well behavior of low-barrier hydrogen bonds. These findings provide deeper insight into the pseudochair conformation of carboxyphosphate, and lead to an improved mechanistic understanding of this intermediate in ATP-dependent carboxylases