Born-Oppenheimer ab initio QM/MM molecular dynamics simulations of the hydrolysis reaction catalyzed by protein arginine deiminase 4

Abstract

Protein arginine deiminase 4 (PAD4) catalyzes the citrullination of the peptidylarginine via two successive stages: deimination and hydrolysis. Herein, by employing state-of-the-art Born-Oppenheimer ab initio QM/MM molecular dynamics simulations with the umbrella sampling method, we characterized the catalytic mechanism of the hydrolysis reaction: first, the nucleophilic attack of a water molecule to the C(zeta) of the thiouronium intermediate yields a stable tetrahedral intermediate, and then the S-C(zeta) bond breaks to generate the final product, citrulline. Throughout the hydrolysis reaction, His471 and Asp473 play pivotal catalytic roles by first enhancing the nucleophilic ability of the active water through forming shorter and low-barrier hydrogen bonds and then by serving as proton-accepting groups to deprotonate the water molecule, which is consistent with experimental findings. At the transition state, the spontaneous proton transfer among the reactive water, His471 and Asp473 have been observed. The determined overall free energy barrier for this hydrolysis stage is 16.5 kcal x mol(-1), which is lower than the barrier of 20.9 kcal x mol(-1) for the deimination stage determined previously with the same computational approach [J. Phys. Chem. B 2009, 113, 12750-12758]. Thus, the rate-determining step of the PAD4-catalyzed citrullination is the first step of the deimination. Our current work further demonstrates the strength and applicability of the ab initio QM/MM MD approach in simulating enzyme reactions.

Figure 1

Previously characterized reaction mechanism for the initial deimination reaction catalyzed by PAD4 [15] (colored in black) and the currently determined reaction mechanism for the second hydrolysis stage (colored in red).

Figure 2

The illustration of the QM/MM partition. Four boundary atoms are colored red and all the other QM atoms are blue. The other atoms belong to the MM subsystem. The reaction center is defined as the C_ζ atom.

Figure 3

Illustration of the structure and the hydrogen bond network of the thiouronium intermediate.

Figure 4

The calculated free energy profile for the the hydrolysis reaction catalyzed by PAD4. The reaction starts from the thiouronium intermediate. The overall reaction consists of two steps: the formation of a tetrahedral intermediate and the cleavage of C_ζ-S bond. The reaction coordinate for the first step was defined as the distance between the OW and C_ζ, while in the second step the breaking bond C_ζ-S is chosen as the reaction coordinate. A total of 30 simulation windows with harmonic potential force constants of 40 ~ 300 kcal·mol^–1 · Å^–2 have been employed. The total length of B3LYP(6-31G*) QM/MM MD simulations to determine this free energy profile is 900 ps.

Figure 5

Illustration of the structures along the hydrolysis reaction. They correspond to the three configurations of the transition state I, and the tetrahedral intermediate, transition state II, product.

Figure 6

Time dependence of the two H-OW bonds for 30 ps QM/MM MD simulation for the window of the TS I. H₁ and H₂ are referred to as the protons hydrogen bonded with His471 and Asp473, respectively.