Chymotrypsin, trypsin, and elastase are three enzymes that cleave protein chains.
First, the structural similarity:
|Superposition of Trypsin (yellow), Elastase (green), and Chymotrypsin (blue) Backbones|
The three amino acids picked out in red are the three that actually do the catalysis.
Here are sequence alignments for chymotrypsin (5cha) and trysin (5ptp), and elastase (1est) and trypsin (1tld) produced by the Swiss PDB Viewer:
|Chymotrypsin vs Trypsin||Elastase vs Trypsin|
What we see here is a clear example of divergent evolution. All are derived from a common ancestral serine protease, and are described as homologous. Taking a step further back, one finds that some non-mammalian serine proteases have 20-50% sequence identity with mammalian ones, suggesting a common ancestral protease an evolutionary step further back.
Evolution can converge on functionality also. The first crystal structure of a bacterial serine protease, subtilisin, from B. amyloliquefaciens, shows a thoroughly different construction from the mammalian ones, and essentially no sequence homology:
|Subtilisin (B. amyloliquefaciens)|
|The orange balls are Ca++, providing thermal stability|
But the enzymes are functionally identical; subtilisin uses the same three catalytic residues, shown in red: Asp32, His64, and Ser221. The mechanism of catalysis is the same, including the positioning of substrate by hydrogen bonding. This appears to be a case of convergent evolution: Mother Nature found a good idea a second time.
As we have seen in several pictures now, the catalytic work of the proteases is done by the so-called catalytic triad, Asp102, His57, and Ser195:
Here is the triad picked out from the crystal structure of chymotrypsin (the extra fine black lines are artifacts of my effort to hide the rest of the enzyme). Remember that X-rays can't see hydrogen atoms; we have to infer their positions.
The mechanism outlined below applies to all of the serine proteases, with small variations. We start with the binding, in cartoon form.
The next step appears to be His57 removing a proton from the the Ser105 OH, while the O does a nucleophilic attack on the peptide carbonyl:
Whether a tetrahedral intermediate is formed was a point of considerable contention in the early investigations of protease mechanisms; biochemists tended to draw nucleophilic acyl substitutions as if they were SN2 reactions - all one step.
This ignored a fundamental difference between the two kinds of reaction.
|LUMO of Ethyl Chloride|
You can see that it involves chiefly the C-Cl bond, and has a considerable back lobe where the nucleophile interacts. Now here is the LUMO of acetyl chloride, CH3(C=O)Cl:
|LUMO of Acetyl Chloride|
This LUMO is almost exclusively on the C=O, and hence there is no way an attacking nucleophile can break the C-Cl bond directly. In short, a tetrahedral intermediate MUST form because the orbital construction of the substrate won't permit any other pathway. End of story.
OK, so we've got the tetrahedral intermediate. Where next? Here's the tetrahedral intermediate, bound to Ser195 with the former carbonyl oxygen in the "oxyanion hole".
The next step is the reconstruction of the carbonyl double bond, with expulsion of the leaving group - in this case, the rest of the protein. This is the stage at which the protein chain actually is cleaved, and it produces an "acyl enzyme": the acyl part of the peptide that was cleaved, bound as an ester to Ser195.
OK, now we've got to cleave the acyl enzyme; enzyme are catalysts, and are not permanently altered in the reaction. To do this, we need a molecule of water.
From here on out, we're writing the mechanism for hydrolysis of an ester:
Restore the carbonyl double bond:
This releases the other end of the original protein, and restores the catalytic triad to its beginning state:
Dissociation of the second protein fragment leaves the enzyme ready to go again.