The mechanism whereby chymotrypsin catalyzes the hydrolysis of a peptide bond can be represented in several steps as shown in the figure below. Each step can be defined as a stationary point on the Potential Energy Surface (PES). These stationary points were calculated using PM7, and can be investigated in detail in the links named "Step n" below. The starting point was 8GCH.pdb. In 8GCH the substrate is the tripeptide Gly250-Ala251-Trp252, with Trp252 terminated by a carboxylic acid group. This sequence also occurs in chymotrypsin at residues 205, 206, and 207, with residue 208 being Thr, so it is reasonable to assume that the substrate is the product of hydrolysis of the Trp-Thr peptide bond in a chymotrypsin by the chymotrypsin in 8GCH. In order to begin modeling the chymotrypsin mechanism, one of the oxygen atoms on carboxylate on Trp252 was replaced by Thr253, this giving a peptide bond ideally positioned for the simulation described here.
All the steps have the same formula, C1115H2450N301O706S16, or 4,588 atoms, so a comparison of the various structures can be made. To reproduce the working shown on this page and those linked to it, download the ARC files for Steps 1 to 6 (on this page) and follow the instructions for locating and refining transition states in proteins.
NOTE: After this work was published, a method for calculating the interaction energy of a ligand non-covalently bound to a protein was developed. This required increasing the accuracy of the geometry optimizations, and resulted in several changes being made to MOPAC. Because of this, there will be significant differences in the various heats of formation cited here, and much smaller differences in derived quantities such as relative energies.
Step 1: Substrate is
docked in active site. This geometry can be arbitrarily defined as the
starting point of the catalytic mechanism. (ARC
Step 2: The Ser195 hydroxyl oxygen bonds to the peptide carbon, and its proton migrates to His57. (carbon, and its proton migrates to His57. (ARC file)
Step 3: The proton on His57 migrates to the nitrogen atom of the peptide bond being hydrolyzed, resulting the the formation of a Zwitterion. The anionic oxygen attached to the tetrahedral carbon is stabilized by hydrogen bonding to the oxyanion hole. (ARC file)
Step 4: The now-unstable Zwitterionic peptide bond breaks, forming an ester and a chain fragment (here Thr253) which then migrates out of the active site. (ARC file)
Step 5: A water molecule adds to the ester to re-form a tetrahedral carbon. The departing Thr253 ionizes. (ARC file)
Step 6: The proton on the tetrahedral carbon migrates to Ser195, splitting the Ser195 - Trp252 ester bond to form an acid plus alcohol. (ARC file)
Step 1: New substrate docks into active site.
Notes: Each step represents a different geometry, i.e., a set of coordinates. The difference between each set of coordinates can be expressed as the sum of the differences in positions of equivalent atoms in the two sets of coordinates. Thus for
The motion in going from one step to the next step looks large, but if even a small bias pulling one geometry in the direction of another is applied, then the "distance" between the geometries would drop considerably.
For six steps, there are 15 = (6·5)/2 possible transition state. Of these, five are "real" in the sense that they represent barriers between adjacent steps, the other ten transition
The transition state between Step 3 and Step 4 is activationless - the Thr353 cation spontaneously migrates out of the active site.
The transition state between Step 2 and Step 4 collapses back to the transition state between Step 1 and Step 2, indicating that this is a nonsense reaction.
The lowest energy pathway is Step 1 → Step 2 → Step 3 → Step 4 → Step 5 → Step 6, i.e., the classical chymotrypsin mechanism.
Energy differences are precise to ~±2 kcal/mol, and are of unknown accuracy. The loss of precision is due to the very flat PES at the local energy minimum. The heats of formation given here represent the fully optimized systems, i.e., stationary points on the PES. Even small changes, such as proton migration from Step 2 to Step 3 result in a a large increase in energy, typically several tens of Kcal/mol, and only after the system relaxes completely are the ΔHf given here obtained.