Chorismate Mutase Reaction using Potential Energy surface scaning

Introduction

The biosynthetic pathway of aromatic amino acids—such as phenylalanine and tyrosine—is critically dependent on the conversion of chorismate to prephenate, a reaction catalyzed by chorismate mutase (Figure 1). This enzyme operates as a homotrimer, with its substrate-binding sites located at the interfaces between subunits. It accelerates the reaction through a concerted mechanism that proceeds without the formation of a covalent intermediate. While the precise role of the enzyme in catalysis remains a topic of active investigation, this aspect will not be explored in detail here. Instead, this tutorial focuses on introducing the application of Potential Energy Surface (PES) scans and quantum chemistry/molecular mechanics (QC/MM) approaches as tools for sampling enzymatically catalyzed reactions.

Our objective here is to focus specifically on PES scanning; therefore, all system preparation steps have been omitted, under the assumption that the user has already completed the preceding basic tutorials on 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:

chorimate mutase pkl file.

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

--------------------------------------------------------------------------------

---------------------- 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.