(Modeling proteins) (Chymotrypsin Mechanism: Step 1, Step 2, Step 3, Step 4, Step 5, Step 6)
Like all catalyic processes, the chymotrypsin mediated hydrolysis of a peptide bond involves a cyclic mechanism - the substrate (the precursor) is bound to the active side, hydrolysis occurs, then the products leave, and finally a new substrate binds to the active site. For convenience, therefore, the start of the process is defined here as the substrate non-covalently bonded to the active site i.e., "docked."
Getting from the original PDB structure to the fully-optimized starting model requires much work. The main steps are: adding hydrogen atoms to the PDB file; optimizing the positions of the hydrogen atoms; forming salt bridges (i.e. ionizing various sites); editing the substrate to make it into a hydrolysable peptide; and unconstrained optimization of the geometry. (PDB file) (ARC file)
(A) The active site, composed of the catalytic triad, the substrate, and Ser214. The hydroxyl hydrogen of Ser214 hydrogen bonds to the carboxylate oxygen of Asp102 that forms a hydrogen bond with the imidazole hydrogen of His57. At the same time, the carbonyl oxygen of Ser214 hydrogen bonds to both the peptide hydrogen of Trp252 and the hydroxyl hydrogen of Ser195, this "locking" the orientation of the hydroxyl hydrogen.
(B) In the oxyanion hole, the carbonyl oxygen of Trp252 forms two normal (1.81Å and 2.02Å) hydrogen bonds with Gly193 and Ser195 peptide hydrogen atoms, respectively.
(C) Chymotrypsin consists of three chains, E, F, and G, with the composition as shown below. There is a fourth chain in 8GCH, chain C, consisting of three residues. To make the substrate, Trp252 was modified to convert the carboxylic acid group into -Thr253. Of all the residues in chymotrypsin, sixteen are of particular interest; these are also shown below. Most of Ser11 is missing; all that survives is the -NH3(+); this forms a salt bridge with Glu20.
| Chain | Amino-acid sequence in Chymotrypsin | Interesting Residues | Chain | Property | |
| E (First residue is Cys1) |
Residues 1 2 3 4 5 6 7 8 9 10
0 CYS GLY VAL PRO ALA ILE GLN PRO VAL LEU+
10 SER
|
Ser11 | E | Salt bridge with Glu20 | |
| Ile16 | F | Salt bridge with Asp194 | |||
| F (First residue is Ile16) |
1 2 3 4 5 6 7 8 9 10
10 ILE+ VAL ASN GLY GLU-
20 GLU- ALA VAL PRO GLY SER TRP PRO TRP GLN
30 VAL SER LEU GLN ASP LYS THR GLY PHE HIS
40 PHE CYS GLY GLY SER LEU ILE ASN GLU ASN
50 TRP VAL VAL THR ALA ALA HIS CYS GLY VAL
60 THR THR SER ASP VAL VAL VAL ALA GLY GLU
70 PHE ASP GLN GLY SER SER SER GLU LYS ILE
80 GLN LYS LEU LYS ILE ALA LYS VAL PHE LYS
90 ASN SER LYS TYR ASN SER LEU THR ILE ASN
100 ASN ASP- ILE THR LEU LEU LYS LEU SER THR
110 ALA ALA SER PHE SER GLN THR VAL SER ALA
120 VAL CYS LEU PRO SER ALA SER ASP ASP PHE
130 ALA ALA GLY THR THR CYS VAL THR THR GLY
140 TRP GLY LEU THR ARG TYR
|
Glu20 | F | Salt bridge with Ser11 | |
| Glu21 | F | Salt bridge with Arg154 | |||
| His57 | F | Catalytic triad | |||
| Glu102 | F | Catalytic triad | |||
| Arg154 | G | Salt bridge with Glu21 | |||
| Ser189 | G | Hydrophobic pocket | |||
| Gly193 | G | H-bond of oxyanion hole | |||
| Asp194 | G | Salt bridge with Ile16 | |||
| Ser195 | G | Catalytic triad | |||
| Ser195 | G | H-bond of oxyanion hole | |||
| Ser195 | G | H-bond to Ser214 | |||
| Ser214 | G | H-bond to Trp252 peptide N | |||
| G (First residue is Asn150) |
1 2 3 4 5 6 7 8 9 10
140 ASN
150 THR PRO ASP ARG+ LEU GLN GLN ALA SER LEU
160 PRO LEU LEU SER ASN THR ASN CYS LYS LYS
170 TYR TRP GLY THR LYS ILE LYS ASP ALA MET
180 ILE CYS ALA GLY ALA SER GLY VAL SER SER
190 CYS MET GLY ASP- SER GLY GLY PRO LEU VAL
200 CYS LYS LYS ASN GLY ALA TRP THR LEU VAL
210 GLY ILE VAL SER TRP GLY SER SER THR CYS
220 SER THR SER THR PRO GLY VAL TYR ALA ARG
230 VAL THR ALA LEU VAL ASN TRP VAL GLN GLN
240 THR LEU ALA ALA ASN |
Gly216 | G | Hydrophobic pocket | |
| Gly216 | G | H-bond to Gly250, Ala 251 | |||
| Ser217 | G | H-bond to Trp252 heterocycle N | |||
| Gly226 | G | End of hydrophobic pocket | |||
Gly250-Ala251-Trp252-Thr253 |
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| C | |||||
| (The substrate) | |||||
|
Fast display or High-quality display Toggle display all Toggle center picture Fit to screen Interesting parts:
one that will hydrolyze the ester between Ser195 and Trp252 Catalytic triad:
Ser214 is very near to Asp102 and Ser195. Its hydroxyl group forms a strong hydrogen bond with Asp102, and its carbonyl oxygen forms a weaker hydrogen bond with the hydroxyl of Ser195, locking the proton in place. Substrate:
Oxyanion hole plus substrate:
The peptide hydrogen atoms of Gly193 and Ser195 form hydrogen bonds with the peptide carbonyl oxygen atom of the bond that is to be hydrolyzed. Substrate in Hydrophobic pocket:
Chymotrypsin specificity is determined by the S1 residues Ser18, Gly216, and Gly226. The substrate forms hydrogen bonds: Ser214 (O of carbonyl) - Trp252 (H of amide) (P1 residue) Gly216 (antiparallel b-sheet - Gly250) P3 Gly193 and His57 to Thr253 Trp215 does not appear to bond to the substrate! Salt bridges:
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