The enzyme chymotrypsin hydrolyzes peptide bonds in proteins. A good starting point for modeling chymotrypsin is the PDB file 8GCH, this contains a complete copy of chymotrypsin plus a tripeptide substrate. This substrate is a product of autolysis, and can be used in constructing a realistic substrate that would be suitable for modeling the hydrolysis mechanism. A description of the computed mechanism is available on-line, and all the files used in that work are available as supplementary material, so only the steps involved in constructing the starting model (step 1 of the mechanism) from 8GCH need be described here.
Before starting, the substrate needs to be modified to make it more realistic. Substrate residue Trp252 terminates in a -COO group, this is clearly the product of the hydrolysis of a peptide bond. Inspection of 8GCH shows that the sequence of residues in the substrate, Gly-Ala-Trp, occurs exactly once, in chain "G" at residues 205, 206, and 207. The next two residues in chain "G" are Thr and Leu. From this sequence, a reasonable conclusion would be that, before hydrolysis occurred, the substrate sequence was Gly-Ala-Trp-Thr-Leu.
The substrate was edited to replace OXT of Trp252 with Thr. To minimize the effect of the carboxylate end of Thr, this residue was edited to add a nitrogen atom to the peptide "C" atom, i.e., to edit -NH-CH(C(C(H)(CH3)(OH))-CHO to -NH-CH(C(C(H)(CH3)(OH))-C(NH2)(O). (The hydrogen atoms are not present at this point, they are only shown here to simplify reading the formulae.) As a result of the extra atoms being added, one water atom HOH487 was deleted and three water oxygen atoms, those for HOH615, HOH625, and HOH647 were moved to avoid steric clashes. All atoms added or moved can be identified in the next data-set by the presence of the text "PROT"
PDB file 8GCH does not include hydrogen atoms, so the first step is to hydrogenate the system. Keywords used in this file are:
ADD_H: Hydrogen atoms are added so as to neutralize all sites. This operation is almost always used together with a SITE keyword which changes the hydrogenation of various sites so that the system is more realistic.
SITE=(salt,"[HIS]57:F.NE2"(0)): SITE adds and deletes hydrogen atoms as necessary to make a realistic system. The two SITE arguments used here are SALT - make all reasonable salt bridges between residues, and "[HIS]57:F.NE2"(0) - SALT had made a salt bridge between Asp102 and His57, but His57 should be neutral, so this option removes a hydrogen atom. Of the two hydrogen atoms in the imidazolium ring, the one that's further from Asp102 is deleted.
HTML: Not essential, but very useful in deciding where to add or delete hydrogen atoms. HTML produces a JSmol web-page that can be opened using NETSCAPE. Individual atoms to be used in the SITE keyword can easily be identified in this web-page. A useful trick is to start editing the SITE keyword and hover the cursor over the appropriate atom in the web-page. That displays the JSmol label, which can then be typed in the SITE keyword. If the PDB label is available, that can be used.
NOCOMMENTS: Suppress all PDB HEADER, TITLE, COMPOUND, SOURCE, etc., remarks.
OUTPUT: Suppress all voluminous output.
NOOPT, OPT-H, MOZYME, CHARGE=-1 EPS=78.4: These keywords are not used in this job, but are present so they will be in the keyword list for the next job.
Run "8GCH Add-H.mop" and copy the resulting ARC file, giving the copy the name "8GCH Opt-H.mop"
Edit "3CSM Add-H.arc" to replace
START_RES=(1A-222 300A-400) chains=(A) HTML output
with
START_RES=(1A-222 300A-400) chains=(A) HTML Opt mozyme eps=78.4 gnorm=5 output charge=0 t=2w
These are the keywords for an unconstrained global optimization of a system in aqueous media. Normally there would be an intermediate step in which the positions of the hydrogen atoms would be optimized, but as this is a very simple reaction, this step is not necessary.
Starting with "3CSM Add-H.arc" use an editor to move the O7 in the substrate away from C5, i.e., deliberately break the C5-O7 bond. Save the resulting structure as "3CSM Chorismate.mop"
Starting with "3CSM Add-H.arc" use an editor to move the C9 in the substrate away from C1, i.e., deliberately break the C9-C1 bond. Save the resulting structure as "3CSM Prephenate.mop"
Run both "3CSM Chorismate.mop" and "3CSM Prephenate.mop" These jobs take a long time, typically a few days, so a useful strategy is to run both of them at the same time on a machine that is dedicated to running long jobs.