Role of Histidine-85 in the Catalytic Mechanism of Thymidine Phosphorylase As Assessed by Targeted Molecular Dynamics Simulations and Quantum Mechanical Calculations

Abstract

The structural changes taking place in the enzyme thymidine phosphorylase (TPase, also known as PD-ECGF) that are required to achieve catalytic competence upon binding thymidine and phosphate have been simulated by means of targeted molecular dynamics (tMD). The hinge regions were characterized by structural homology comparisons with pyrimidine nucleoside phosphorylase, whose X-ray structure has been solved both in a closed and in an open form. The rearrangement of residues around the substrate that was observed during the tMD trajectory suggested that His-85 could be playing an important role in the catalytic mechanism. A quantum mechanical study of the reaction in the presence of the most relevant active site residues was then performed at the semiempirical level. The results revealed that His-85 could be involved in the protonation of the pyrimidine base at the O2 position to yield the enol tautomer of the base. To establish the role of this oxygen atom in the reaction, ground states, transition states, and final products were studied using higher level ab initio methods starting from both thymidine and 2-thiothymidine as alternative substrates. Comparison of both transition states showed that replacing the oxygen at position 2 of the pyrimidine base by sulfur should accelerate the reaction rate. Consistent with this result, 2-thiothymidine was shown to be a better substrate for TPase than the natural substrate, thymidine. For simulating the final step of the reaction, tMD simulations were used to study domain opening upon product formation considering both the enol and keto tautomers of thymine. Product release from the enzyme was easiest in the simulation that incorporated the keto tautomer of thymine, suggesting that the enol intermediate spontaneously tautomerizes back to the more energetically stable keto form. These results highlight a previously unreported role for His-85 in the catalytic mechanism of TPase and can have important implications for the design of novel TPase inhibitors.

The Nucleoside Derivative 5′-O-Trityl-inosine (KIN59) Suppresses Thymidine Phosphorylase-triggered Angiogenesis via a Noncompetitive Mechanism of Action

“…It is assumed that a domain closure of the cleft is necessary to generate the active site of the enzyme. This domain closure has been simulated by molecular modeling studies and the catalytic mechanism of the enzyme has been proposed (36,37). Very recently, the structure of the human TPase has been revealed in complex with a pyrimidine derivative, TPI that appears to mimic the substrate transition state (15).…”

Identification of aspartic acid-203 in human thymidine phosphorylase as an important residue for both catalysis and non-competitive inhibition by the small molecule “crystallization chaperone” 5′-O-tritylinosine (KIN59)

“…The missing bonded and nonbonded parameters were assigned, by analogy or through interpolation from those already present in the AMBER database, in a way consistent with the AMBER force field [32]. The geometry and atom point charges for the transition state (TS) of the phosphorolysis reaction were retrieved from previous work [33].…”

“…A model of D203A TP was constructed using the mutagenesis module available in the molecular graphics program PyMOL (http://www.pymol.org/).The complexes of wild-type and D203A TP with the transition state (TS) of the reaction [33] and of wild-type TP with KIN59 were neutralized by addition of sodium ions [35] and immersed in truncated octahedrons of TIP3P water molecules [36]. Periodic boundary conditions were applied and electrostatic interactions were treated using the smooth particle mesh Ewald method [37] with a grid spacing of 1 Å .…”

“…We therefore decided to further focus on some prior conformational state(s) of the enzyme for our docking studies. Since open and closed TP conformations are connected by hinge-dependent interdomain motions [33], as is also the case in the related pyrimidine nucleoside phosphorylase (PyNP) [49], we used normal mode analysis (NMA) to reproduce this motion and generate a series of intermediate protein conformers that could help in the identification of a region capable of lodging the hydrophobic and polar substituents of 5 0 -O-tritylinosine.…”

“…Time evolution of the overall interaction energy between the transition state of the reaction and either wild-type (blue) or D203A (magenta) human TP during the molecular dynamics simulations. reaction transition state (TS) has been previously characterized in detail [33], unrestrained MD simulations of each enzyme in the presence of an active site-bound TS were undertaken to analyze the relative binding affinities in the two complexes.…”