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Transimination Reaction at the Active Site of Aspartate Aminotransferase: A Proton Hopping Mechanism through Pyridoxal 5'-Phosphate Kumari Soniya, Shalini Awasthi, Nisanth N. Nair, and Amalendu Chandra ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.9b00834 • Publication Date (Web): 30 May 2019 Downloaded from http://pubs.acs.org on May 30, 2019
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ACS Catalysis
Transimination Reaction at the Active Site of Aspartate Aminotransferase: A Proton Hopping Mechanism through Pyridoxal 50 -Phosphate Kumari Soniya, Shalini Awasthi† , Nisanth N. Nair∗ and Amalendu Chandra∗ Department of Chemistry, Indian Institute of Technology Kanpur, Uttar Pradesh, India 208016 ∗
For correspondence.
E-mail:
[email protected], Tel: +91 512 2597241 (AC);
[email protected], Tel: +91 512 2596311 (NNN) †
Current address: Warwick Manufacturing Group, University of Warwick, Coventry,
United Kingdom, CV47AL.
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Abstract Transimination reaction involves conversion of an internal aldimine involving pyridoxal 50 -phosphate (PLP) and an enzyme to an external aldimine involving PLP and a substrate amino acid and it constitutes an essential step in many biological processes catalyzed by PLP-dependent enzymes. We have investigated the free energy landscape and mechanistic pathways of transimination process at the active site of aspartate aminotransferase by means of hybrid quantum-classical molecular dynamics simulations combined with various enhanced sampling techniques. It is found that, after a geminal diamine is formed in the first step of the process, the reaction proceeds through a path where a proton from the amine nitrogen of the substrate amino acid is transferred first to the phenolic oxygen of the PLP ring, and from there it gets transferred to the imine nitrogen of the active site lysine in the next step of the reaction. Both these proton transfer events are found to be assisted by relative rotation of the PLP ring which brings the phenolic oxygen of PLP closer to the amine and imine nitrogens of the substrate and lysine, respectively. The transfer of proton from the phenolic oxygen of PLP to the active site lysine residue is found to be the rate determining step with an effective barrier of only 4 kcal/mol. Neither any direct proton transfer from lysine to the substrate nor any indirect proton transfer involving any active site residue or water is found.
Keywords: Transimination reaction; Aspartate Aminotransferase; QM/MM Simulation, Metadynamics, Temperature Accelerated Sliced Sampling
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Introduction Transaminase or aminotransferase enzymes are responsible for the conversion
of amino acids to keto acids by the transamination process. The catalytic activities of these enzymes are supported by the pyridoxal 50 -phosphate (PLP) coenzyme. PLP is the biologically active form of vitamin B6 and it acts as a coenzyme in several enzymatic processes apart from transamination 1–3 . In biological systems, PLP is found to be covalently bound as a Schiff base with the side chain nitrogen atom of the active site lysine residue of the enzyme (called the internal aldimine or IA) or to the amine group of substrate amino acids (called the external aldimine or EA). Its ability to form Schiff bases has made PLP a versatile cofactor for various types of PLP-dependent enzymes. Presently, PLP dependent enzymes are classified into five categories depending upon the overall fold of the enzyme 4 . The most fascinating attribute common of all the PLP dependent enzymes is the conversion of internal aldimines to external aldimines via the process of transimination (Figure 1). The subsequent pathways of the catalytic action of PLP dependent enzymes vary. 1,2 There have been a number of theoretical and experimental studies to investigate the reaction mechanism of transimination 5–7 , but the mechanistic pathway is still not fully understood for this important reaction. The latest hypothesis is that the reaction takes place via the formation of two geminal diamine intermediates (GDI1 and GDI2) which differ with the position of a proton 5–8 . Further, the mechanistic aspects of the conversion of GDI1 to GDI2 through proton transfer has also remained an unresolved issue in the transimination process 9–13 . Possible suggested pathways for this proton transfer step are shown in Figure 2. In the current study, we have looked at the detailed pathway of the transimination process by means of hybrid quantum-classical molecular dynamics simulations combined with various enhanced sampling techniques. In particular, we have considered one of the transaminase enzymes, namely the aspartate aminotransferase (AspAT), that is active in the amino acid catabolism pathway for aspartic acid.
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AspAT is involved in the conversion of aspartate and alpha ketoglutarate to oxaloacetate and glutamate. It belongs to the Type 1 PLP-dependent enzymes and is one of the most studied enzymes of this category. It exists as a homodimeric unit in nature with each monomer having a small and a large domain. AspAT is found in liver, cardiac muscle, skeletal muscle, kidneys, brain, and red blood cells. In the internal aldimine form, the coenzyme (PLP) is bound to the active site lysine residue of AspAT. This bond is replaced by a bond with the substrate aspartic acid to give the external aldimine via the transimination process (Figure 1). Availability of good resolution X-ray structure of AspAT has made it possible to use it as a model system for studying the mechanistic pathway of transimination reaction. Here, we have studied the transimination process at the active site of AspAT through hybrid quantum-mechanical/molecular-mechanical (QM/MM) molecular dynamics simulations combined with various enhanced sampling techniques. The computational details are provided in the next section.
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Computational Details The transimination step was studied starting from the Michaelis complex (IA).
The structure of IA and the substrate aspartic acid was built using the coordinates of the dimeric AspAT from the X-ray crystal structure (PDB ID: 1AHY) 14 . The protein structure was then solvated by 29,746 TIP3P 15 water molecules in a rectangular box with dimension of 104.15× 113.48× 85.56 Å3 using the LEaP program in AMBER 16 and the corresponding parameter files were generated. Hydrogen atoms were added considering a pH of 7.4. It is believed that one hydrogen from the cationic amine group of ASP substrate is transferred to the imine nitrogen of PLP-LYS internal aldimine to form the Michaelis complex (IA) 17,18 . Therefore, the carboxylic groups of the substrate aspartic acid were taken in conjugate base form while the amine group is kept neutral. The reaction should proceed with the ketoenamine state of PLP which is the more stable tautomer at the active site of the enzyme 19–21 . Therefore, the NZ atom is kept protonated and the phenolic oxygen (O3A) carries a negative charge which gets delocalized over the PLP ring (Figure 1). The pyridine ACS Paragon Plus Environment
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nitrogen (N1) (see Figure 1) is kept protonated based upon earlier studies where it was shown that the pyridine nitrogen in aspartate aminotransferase was protonated prior to the reaction. 22–24 . We also performed two static quantum chemical calculations using ONIOM method 25 for the entire enzyme-PLP complex to determine the starting protonation state of N1 in PLP. Details of the ONIOM calculations are provided in the Supporting Information (SI) and the results are included in Table S1. The protonated state of N1 of PLP is found to be more stable compared to the state where N1 is unprotonated. Hence, in our subsequent simulations of the transimination reaction, we have taken N1 of PLP in its protonated form. The coenzyme, PLP, was parameterised using the generalized amber force field (GAFF) 26 and potential of the rest of the protein was obtained using the ff99SB 27 force field. 24 Na+ ions were added to the system for charge neutralization. Energy minimization and equilibration of the system were done using the sander module of AMBER program with a time step of 1 fs. The system was equilibrated in N P T ensemble for 500 ps at 300 K and 1 atm pressure using Berendsen barostat 28 . Subsequently, the production run was carried out in N V T ensemble for 5 ns. Temperature was maintained at 300 K using Langevin thermostat. The classically equilibrated system was then re-equilibrated in QM/MM framework 29,30 . The PLP, active site Lys258 residue, substrate ASP and the nearby Tyr0 70 residue were treated quantum mechanically in the QM/MM calculations; see Figure 3. The active site Tyr0 70 residue resides near the aldimine which could assist in the proton transfer step from GDI1 to GDI2. The bonds between CA and CB atoms of both Tyr0 70 and Lys258 residues were cut and the dangling bonds were capped using hydrogen atoms (Figure 3). The QM box length was about 22 Å. The rest of the protein residues not directly participating in the reaction and also the water molecules were treated classically using the CPMD/GROMOS96 interface 31 to minimize the computational cost. The QM/MM calculations were done using the BLYP functional and plane wave basis set with kinetic energy cut off of 70 Ry. Kleinman-Bylander pseudopotentials were used. The electronic degrees of freedom were given a fictitious mass of 700 a.u. to maintain adiabaticity in the QM region with a time step of 6 a.u. The ACS Paragon Plus Environment
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QM and MM subsystems were thermostated by separate Nosé–Hoover chain thermostats 32,33 . Enhanced sampling techniques, namely the temperature accelerated sliced sampling (TASS) 34 and well-tempered metadynamics 35 , were used to study the transimination reaction at the active site of AspAT. In these simulations, we considered n number of collective variables (CVs) s ≡ {s1 , · · · , sn } which are relevant to represent the free energy surface and are necessary to enhance the sampling of conformational changes. In well-tempered metadynamics simulations, a time-dependent bias potential constructed by sum of Gaussian biases is deposited at discrete times along the trajectory of the CVs as 35 (s − sτ )2 , V (s, t) = wτ exp − 2 (δs)2 τ
