Free Energy Landscapes of the Tautomeric Interconversion of Pyridoxal 5′-Phosphate Aldimines at the Active Site of Ornithine Decarboxylase in Aqueous Media

Abstract

The pyridoxal 5′-phosphate (PLP) acts as a coenzyme for a large number of biochemical reactions. It exists in mainly two bound forms at the active site of the concerned enzyme: the internal aldimine, in which the PLP is bound with the ϵ-amino group of lysine at the active site, and the external aldimine, where the PLP is bound to the substrate amino acid. Both the internal and external aldimines have Schiff base linkage (N−H−O) and can exist in two tautomeric structures of ketoenamine and enolimine forms. In this work, we have investigated the free energy landscape for the tautomeric proton transfer in the internal and external aldimines at the active site of the ornithine decarboxylase enzyme in an aqueous medium. We performed hybrid quantum-classical metadynamics and force field-based molecular dynamics simulations, which revealed that the ketoenamine tautomer is more stable than the enolimine form. The QM/MM metadynamics calculations show that the free energy difference between the ketoenamine and enolimine forms for the internal aldimine is 3.9 kcal/mol, and it is found to be 5.8 kcal/mol for the external aldimine, with the ketoenamine form being more stable in both cases. The results are further supported by calculations of the binding free energies from classical simulations and static quantum chemical calculations in different environments. We have also analyzed the configurational structure of the microenvironment at the active site in order to have better insights into the interactions of the active site residues with the PLP in its two tautomeric forms.

1,3-Proton Transfer of Pyridoxal 5′-Phosphate Schiff Base in the Branched-Chain Aminotransferase: Concerted or Stepwise Mechanism?

“…This observation suggests that although Lys204 acts as a general base to abstract the proton, the protonated amino group becomes unstable due to the proximity of the carboxyl group. This phenomenon of the carboxyl group capturing surrounding protons has also been observed in ornithine decarboxylase . In Int, there is a change in the hybridization of Cα from sp 3 (in R) to sp 2 .…”

“…In the present study, the calculations begin with the external aldimine form of PLP as it is the prerequisite for catalysis in PLP-dependent enzymes, and its crystal structure is readily available. PLP exhibits two tautomers resulting from intramolecular proton transfer: the O-protonated covalent enol-imine form and the N-protonated zwitterionic keto-enamine form . Among various PLP-dependent enzymes, the keto-enamine tautomer is the predominant conformation observed in enzymes such as aspartate aminotransferase, Ala-racemase, and O-acetylserine sulfhydrylase. , However, there are a few exceptions where the enol-imine tautomer is favored, as seen in ornithine 4,5-aminomutase .…”

“…This phenomenon of the carboxyl group capturing surrounding protons has also been observed in ornithine decarboxylase. 53 In Int, there is a change in the hybridization of Cα from sp 3 (in R) to sp 2 . This change in hybridization is accompanied by a decrease in the Cα-N2 bond length from 1.49 to 1.32 Å. Additionally, the C4−C4′ bond length decreases from 1.47 Å (in R) to 1.42 Å due to the resonance effect.…”