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 BornOppenheimer ab initio QM/MM molecular dynamics simulations with umbrella sampling method, we have characterized the catalytic mechanism of the hydrolysis reaction: first the nucleophilic attack of a water molecule to the C ζ of the thiouronium intermediate yields a stable tetrahedral intermediate, and then the S-C ζ 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 has been observed. The determined overall free energy barrier for this hydrolysis stage is 16.5 kcal·mol -1 , which is lower than the barrier of 20.9 kcal·mol -1 for the deimination stage determined previously with the same computational approach [J. Phys. Chem. B, 113, 12750-12758 (2009)]. 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.

Ab Initio QM/MM Free-Energy Studies of Arginine Deiminase Catalysis: The Protonation State of the Cys Nucleophile

“…The significant difference between the calculated (6.7 kcal/mol) and measured free-energy activation energies argues strongly against the involvement of the deprotonated cysteine nucleophile. A neutral Cys residue in the resting state of ADI is also consistent with our earlier PAD4 study, in which the ionized active site repels the substrate 47. Interestingly, the nucleophilic addition barriers for both free and peptidyl arginines are almost the same in our theoretical calculations (20.9 and 21.3 kcal/mol for PAD4 and ADI).…”

“…Free-energy simulations, particularly those with an ab initio description of the QM region, are necessary to reach quantitatively reliable conclusions 41, 45. Such free-energy QM/MM studies have indeed been performed recently for the catalysis of another GMSF enzyme, PAD4 46-47. To this end, MD calculations indicated that the Michaelis complex with a neutral Cys nucleophile is more stable than that with a deprotonated Cys, and the former yielded a free energy of activation comparable to experimental data 46.…”

The protein arginine deiminases: Structure, function, inhibition, and disease

“…8,29,31 While the initial body of work focused on PAD4, mechanistic studies on PADs 1 and 3 have confirmed that they too proceed through a similar reverse protonation mechanism. 30 Although modeling QM/MM studies 33,34 have suggested that the nucleophilic species is the thiol form of Cys645, it is hard to reconcile this data with the inverse solvent isotope effect observed on k cat / K m with PAD4, as well as PADs 1 and 3. 29,31 Based on these mechanistic studies, as well as mechanistic studies with F-amidine, Cl-amidine, 2-fluoracetamidine, and 2-chloroacetamidine, which show bell shaped pH inactivation rate profiles, we recently proposed that inactivation by F-amidine, Cl-amidine, and 2-fluoroacetamidine occurs via the initial attack of the Cys645 thiolate on the amidinium carbon which results in the formation of a stable protonated tetrahedral intermediate that mimics the initial tetrahedral intermediate formed during substrate hydrolysis (Figure 2); His471 is the likely proton donor.…”

Mechanistic similarity and diversity among the guanidine-modifying members of the pentein superfamily

“…In this model, deprotonation of the core Cys occurs in concert with nucleophilic attack on the guanidine carbon. Computational studies with PAD4 have suggested that a protonated Cys is energetically more favorable than a Cys thiolate in the Michaelis complex [38,39]. In addition, the core Cys residue in AGAT is also proposed to be predominantly protonated in the resting state, possibly activated by Asp305 for attack [33].…”