Chorismate Mutase Reaction using Potential Energy surface scaning
Introduction
Chorismate mutase is an enzyme that catalyzes the conversion of chorismate to prephenate (Figure 1), a crucial step in the synthesis pathway of aromatic amino acids, such as phenylalanine and tyrosine. The enzyme exists as a homotrimer, and the substrate binding sites are located at the interfaces between subunits. The catalyzed reaction takes place in a single step, without the formation of a covalent intermediate with the enzyme. Although there is a broad discussion about the enzyme's major role in the catalysis process, we won't delve into that discussion at this moment. This tutorial aims to introduce the use of Potential Energy Surface Scans (PES) and QC/MM as a method for sampling catalyzed reactions.
Our objective here is to concentrate on PES scanning; as such, all steps related to preparing the system have been omitted, assuming that the user has already completed the basic tutorials related to the subject.
Figure 1: Conversion of chorismate into prephenate. Adapted from Jianpeng and collaborators (https://www.pnas.org/doi/10.1073/pnas.95.25.14640)
System Loading
First, load the system containing the CM enzyme and substrate into EasyHybrid (Figure 2). The previously prepared system is available in the file provided below:
To open this file, navigate to: Main Menu > File > Open (Change the file type from ".easy" to ".pkl"). For more details check the user guide.
Figure 2: Enzyme + substrate complex loaded into EasyHybrid. The region previously determined to be quantum is presented in the form of balls and sticks.
The system was constructed using the amber99SB force field and comprises 8061 atoms, with 5673 being frozen (atoms located outside a 14-angstrom sphere centered on the C5 carbon atom of the substrate). The quantum region consists of 24 atoms (the chorismate molecule) and is described by the semi-empirical Hamiltonian PM6. For the sake of expeditious completion of the tutorial, we opted for the smallest possible quantum region.
Applying of PES Method
Potential energy surface scans are conducted based on the selection of a reaction coordinate. There isn't a single way to define it, but certain choices prove more convenient than others (often, determining the appropriate reaction coordinate is the most challenging step). In our case, the reaction of interest entails the breaking of a chemical bond (C5 – O7, represented by distance d1) and the formation of another (C1 – C9, represented by distance d2), involving four atoms. Therefore, we define our reaction coordinate as the difference between the distances d1 and d2. Figure 3 illustrates the atoms involved in selecting the reaction coordinate.
Figure 3: RC definition, selections #1 and #2 define d1, and selections #3 and #4 define d2.
To open the PES scan window, either click on the PES icon in Figure 4, situated on the toolbar of the main window, or navigate to the main window menu and select 'Simulation > Reaction Coordinate Scans.
Figure 4: The PES icon situated on the toolbar of the main window, or navigate to the main window menu and select 'Simulation > Reaction Coordinate Scans.
In 'picking mode,' choose the atoms as illustrated in Figure 5. In this case, selections #1 and #2 define d1, while selections #3 and #4 define d2. For this tutorial, we will employ 30 steps, each with a displacement of 0.1 angstroms, and a restraining force of 4000 N/mol. All the settings utilized in this step are visible in Figure X. To delve deeper into the functioning of PES scans, refer to this article in our user guide."
Figure 5: PES scan window.
Results and Analysis
This simulation should only take a few minutes on a typical computer. Upon completion, you will obtain a trajectory in the standard pDynamo format (a directory containing coordinate files) and a text file named 'output.log,' which includes the primary simulation data. Below, we provide an overview of how the data is stored in the log file.
TYPE EasyHybrid-SCAN
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---------------------- Coordinate 1 - multiple-Distance ------------------------
ATOM1 = 2034 ATOM NAME1 = C5
ATOM2 = 2037 ATOM NAME2 = O7
ATOM3 = 2039 ATOM NAME3 = C9
ATOM4 = 2026 ATOM NAME4 = C1
NUMBER OF STEPS = 32 FORCE CONSTANT = 4000
DMINIMUM = -1.67527 MAX INTERACTIONS = 600
STEP SIZE = 0.1000000 RMS GRAD = 0.1100000
--------------------------------------------------------------------------------
--------------------------------------------------------------------------------
Frame dist-ATOM1-ATOM2 dist-ATOM3-ATOM4 Energy
--------------------------------------------------------------------------------
DATA 0 1.476385655453 3.151653287235 -24404.518504663480
aDATA 1 1.483052797433 3.059597730188 -24403.987022739653
DATA 2 1.490717937673 2.969218928936 -24402.187171688085
DATA 3 1.499282970927 2.879134992640 -24399.577475320093
DATA 4 1.510351305338 2.792280123376 -24395.137281599305
DATA 5 1.521194614281 2.705154642098 -24389.208864260054
DATA 6 1.533478286380 2.618726206724 -24382.980763339277
DATA 7 1.549909408614 2.537675447706 -24374.112982965780
DATA 8 1.568772814765 2.458281685017 -24363.498109232100
DATA 9 1.590880353879 2.382221011634 -24351.584884703221
DATA 10 1.616705996521 2.309482973565 -24338.218554320138
DATA 11 1.646113082391 2.238402435236 -24325.520297864503
DATA 12 1.682453257551 2.173503746100 -24312.201726441010
DATA 13 1.724210184635 2.110467670474 -24301.173107572005
DATA 14 1.771170624822 2.047496710368 -24295.985435493487
DATA 15 1.797474910004 1.960212282548 -24300.804275409926
DATA 16 1.822537862963 1.876109689300 -24315.024872143567
DATA 17 1.869513281063 1.820105905378 -24333.748622765161
DATA 18 1.923524635895 1.772563337677 -24354.896790823816
DATA 19 1.983479487653 1.733994427407 -24375.460757403638
DATA 20 2.048925043226 1.701190620433 -24394.930846216848
DATA 21 2.119222974368 1.674032333572 -24412.449122955124
DATA 22 2.191888030741 1.649125571382 -24428.792474388694
DATA 23 2.268940242991 1.628967452981 -24442.023790143332
DATA 24 2.350444891422 1.613511480127 -24452.973523875276
DATA 25 2.435124239699 1.600468991387 -24461.830635635015
DATA 26 2.522540149703 1.589887595370 -24469.048267578553
DATA 27 2.611520585558 1.580860247932 -24474.541587330656
DATA 28 2.702713026270 1.573676417209 -24478.702016640047
DATA 29 2.799121945740 1.570645845310 -24483.129495810274
DATA 30 2.892039373227 1.564677738424 -24485.696656509492
DATA 31 2.986195418318 1.560086067528 -24487.323127226369
To interactively view the results, import the trajectory in the 'pkl folder – pDynamo trajectory' format (Figure 6).
Figure 6: Importing data.
Utilize the PES analysis window, which can be found in the main menu under 'Analysis > PES analysis' (Figure 7).
Figure 7: Trajectory inspection and energy profile.