Research Article Cite This: ACS Catal. 2019, 9, 6276−6283
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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* Department of Chemistry, Indian Institute of Technology Kanpur, Kanpur, Uttar Pradesh 208016, India
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S Supporting Information *
ABSTRACT: The transimination reaction involves conversion of an internal aldimine involving pyridoxal 5′-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 the 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 is transferred to the imine nitrogen of the active site lysine in the next step of the reaction. Both of 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 the proton from the phenolic oxygen of PLP to the active site lysine residue is found to be the ratedetermining 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
1. 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 5′-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, PLPdependent enzymes are classified into five categories depending upon the overall fold of the enzyme.4 The most fascinating attribute common of all the PLPdependent 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 PLPdependent 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 © 2019 American Chemical Society
Figure 1. Transimination reaction at the active site of aspartate aminotransferase. Naming of atoms in the text is used as shown in the figure. IA and EA represent the internal aldimine and external aldimine, respectively.
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, Received: February 25, 2019 Revised: May 28, 2019 Published: May 30, 2019 6276
DOI: 10.1021/acscatal.9b00834 ACS Catal. 2019, 9, 6276−6283
Research Article
ACS Catalysis
Figure 2. Different suggested pathways for the proton transfer step from the substrate aspartic acid to Lys258 residue in AspAT for the conversion of GDI1 to GDI2. (a) Indirect proton transfer via some active site residue or water molecule. (b) Direct proton transfer.
2. 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 TIP3P15 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 the 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 becomes delocalized over the PLP ring (Figure 1). The pyridine nitrogen (N1) (see Figure 1) is kept protonated on the basis of 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 the ONIOM method25 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
the mechanistic aspects of the conversion of GDI1 to GDI2 through proton transfer have 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. AspAT is involved in the conversion of aspartate and αketoglutarate 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 the 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). The availability of a good resolution X-ray structure of AspAT has made it possible to use it as a model system for studying the mechanistic pathway of the 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. 6277
DOI: 10.1021/acscatal.9b00834 ACS Catal. 2019, 9, 6276−6283
Research Article
ACS Catalysis
In well-tempered metadynamics simulations, a time-dependent bias potential constructed by the sum of Gaussian biases is deposited at discrete times along the trajectory of the CVs as35 ÅÄÅ ÑÉ Å (s − sτ )2 ÑÑÑ ÑÑ V b(s, t ) = ∑ wτ expÅÅÅÅ− ÅÅ 2(δs)2 ÑÑÑ (1) Ç Ö τ
