Mechanism of Guanosine Triphosphate Hydrolysis by the Visual Proteins Arl3-RP2: Free Energy Reaction Profiles Computed with Ab Initio Type QM/MM Potentials

Abstract

We report the results of calculations of the Gibbs energy profiles of the guanosine triphosphate (GTP) hydrolysis by the Arl3-RP2 protein complex using molecular dynamics (MD) simulations with ab initio type QM/MM potentials. The chemical reaction of GTP hydrolysis to guanosine diphosphate (GDP) and inorganic phosphate (Pi) is catalyzed by GTPases, the enzymes, which are responsible for signal transduction in live cells. A small GTPase Arl3, catalyzing the GTP → GDP reaction in complex with the activating protein RP2, constitute an essential part of the human vision cycle. To simulate the reaction mechanism, a model system is constructed by motifs of the crystal structure of the Arl3-RP2 complexed with a substrate analog. After selection of reaction coordinates, energy profiles for elementary steps along the reaction pathway GTP + H2O → GDP + Pi are computed using the umbrella sampling and umbrella integration procedures. QM/MM MD calculations are carried out, interfacing the molecular dynamics program NAMD and the quantum chemistry program TeraChem. Ab initio type QM(DFT)/MM potentials are computed with atom-centered basis sets 6-31G** and two hybrid functionals (PBE0-D3 and ωB97x-D3) of the density functional theory, describing a large QM subsystem. Results of these simulations of the reaction mechanism are compared to those obtained with QM/MM calculations on the potential energy surface using a similar description of the QM part. We find that both approaches, QM/MM and QM/MM MD, support the mechanism of GTP hydrolysis by GTPases, according to which the catalytic glutamine side chain (Gln71, in this system) actively participates in the reaction. Both approaches distinguish two parts of the reaction: the cleavage of the phosphorus-oxygen bond in GTP coupled with the formation of Pi, and the enzyme regeneration. Newly performed QM/MM MD simulations confirmed the profile predicted in the QM/MM minimum energy calculations, called here the pathway-I, and corrected its relief at the first elementary step from the enzyme–substrate complex. The QM/MM MD simulations also revealed another mechanism at the part of enzyme regeneration leading to pathway-II. Pathway-II is more consistent with the experimental kinetic data of the wild-type complex Arl3-RP2, whereas pathway-I explains the role of the mutation Glu138Gly in RP2 slowing down the hydrolysis rate.

Interplay between the Enamine and Imine Forms of the Hydrolyzed Imipenem in the Active Sites of Metallo-β-lactamases and in Water Solution

“…Benchmark studies demonstrate that conformational dynamics mostly affects the first reaction step as the system in the ES complex area is more flexible compared with the following minima regions. 54 We compare energy profiles for the first step of the imipenem hydrolysis reaction by NDM-1 obtained on the potential energy surface in this study and on the Gibbs energy surface 17 at the same QM(PBE0-D3/6-31G**)/MM theory level. The energy barriers differ 1 kcal/mol being 13 kcal/mol on the potential energy surface and 14 kcal/mol on the Gibbs energy surface.…”

“…Another important issue that is discussed in the QM/MM simulations of enzymatic reactions is the influence of the explicit account for the dynamics of the active site that can be done using umbrella sampling simulations. Benchmark studies demonstrate that conformational dynamics mostly affects the first reaction step as the system in the ES complex area is more flexible compared with the following minima regions . We compare energy profiles for the first step of the imipenem hydrolysis reaction by NDM-1 obtained on the potential energy surface in this study and on the Gibbs energy surface at the same QM­(PBE0-D3/6-31G**)/MM theory level.…”

Two Sides of Quantum-Based Modeling of Enzyme-Catalyzed Reactions: Mechanistic and Electronic Structure Aspects of the Hydrolysis by Glutamate Carboxypeptidase

“…In the first case, the catalytic glutamic acid is replaced by the glutamine residue. At the first glance, this should completely abolish the reaction; however, it is known from previous computational studies that glutamine can act as a proton shuttle [27,28,[47][48][49]. In fact, performance of the E424Q variant in the NAAG hydrolysis is observed experimentally showing only eight times decrease of the V max value as compared with the wild type enzyme [16].…”

“…When computing MD trajectories of about 10 ps length per window, it is possible to apply the umbrella sampling (US) scan of the Gibbs energy surface with the subsequent statistical analysis, using umbrella integration (UI) methods [22,23] to quantify elementary steps of chemical reactions in the enzyme active sites (see, for example, Refs. [24][25][26][27][28]). Using this technique, we are able to construct reaction energy profiles with realistic barrier heights on the reaction pathway.…”

Computational Characterization of N-acetylaspartylglutamate Synthetase: From the Protein Primary Sequence to Plausible Catalytic Mechanism

“…High-performance computing plays an increasingly important role in life sciences, including simulations of chemical reactions in enzymes using advanced modeling methods based on the quantum mechanics/molecular mechanics (QM/MM) theory [1,13,14]. A practical goal of these simulations is to exploit the obtained information on structures and dynamics in protein systems for prediction of novel prospective drugs to fight human diseases [2].…”