Structural Biochemistry/Enzyme/Catalytic Triad

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Catalytic Triad in the Chymotrypsin

A catalytic triad is a group of three amino acids that are found in the active sites of some proteases involved in catalysis.
Three different proteases that have catalytic triads are: chymotrypsin, trypsin and elastase. In chymotrypsin, the catalytic triad is made from serine 195, histidine 57, and aspartate 102. The side chain of serine is bonded to the imidazole ring of the histidine residue which accepts a proton from serine to form a strong alkoxide nucleophile in the presence of a substrate for attack. The aspartate residue orients histidine to make it a better proton acceptor via hydrogen bonding and electrostatic reactions.

The active site of chymotrypsin is marked by serine 195. Serine lies in a small pocket on the surface of the enzyme. Serine is bonded to histidine 57 which is then bound to aspartate 102. All three of these residues are hydrogen bonded at this pocket. These three residues participate in concerted mechanisms that allows chymotrypsin and other proteases to be activated by incoming substrates. This is called the catalytic triad.

We know that serine is the final reactive site but serine actually depends on the histidine and asparate residue to make it a good nucleophile. The histidine residue forces serine into a position that facilitates nucleophilic attack later on through the process of catalysis by approximation. In the presence of a substrate, a chain reaction occurs. First since asparate is acidic, it will be deprotonated first by bases. Aspartate that flanks the histidine residue also provides it with favorable electrostatic effects and makes it a better proton acceptor. So after asparate is deprotonated, proton transfer from histidine goes to aspartate. Now that histidine is deprotonated, it grabs the proton from serine's hydroxyl group. This creates a much more reactive alkoxide group on serine.

Now that the serine is activated we can proceed onto peptide hydrolysis. The alkoxide can attack an incoming substrate to form a tetrahedral intermediate. In this stage we form a resonating oxyanion hole which is a common motif in these kind of reactions. The oxyanion hole stabilizes the tetrahedral intermediate by distributing the negative charge around. Next comes the acyl-enzyme and eventually we see the release of the amine component and water binding.

The catalytic triad actually reveals a deep hydrophobic pocket where serine is sticking out in the center. This pocket positions incoming side chains of a substrate. There is a lot of specificty involved as chymotrypsin has a specific pocket with serine while other enzymes such as trypsin and elastase have different composition of pockets. Therefore we can now know that chymotrypsin likes large aromatic or long, nonpolar side chain.

Catalytic triads also exist in trypsin and elastase. Instead of serine, in trypsin, the center amino acid at the pocket is aspartate. Therefore, its pocket is specific to positively charged species of side chains. Elastase has a pocket that contains two residues of valine, which makes it very hard for big bulky side chains to enter the pocket; therefore, it favors small side chains. Trypsin and elastase are obviously homologs of chymotrypsin. They have 40% similarity in composition and have similar structures.

Reaction Steps of Substrate binding with Catalytic Triad

Site-Directed Mutagenesis helps us understand the Catalytic Triad

Site-directed mutagenesis can be used to test the involvement of individual amino acid residues to the catalytic influence of a protease. Each of the triad’s residues has been converted to alanine. The cleaving ability of each mutant enzyme is examined. The conversion of active-site serine 221, aspartate 32, and histidine 64 into alanine reduces catalytic power. These results strongly support the fact that the catalytic triad, especially the serine-histodine pair, act together to generate a nucleophile that attacks the carbonyl carbon atom of a peptide bond. Site-directed mutagenesis can also tell us the importance of the oxyanion hole for catalysis. The mutation of asparagine 155 to glycine removes the side chain NH group reduced by the oxyanion hole. This shows that the NH group of asparagine residue helps to stabilize the tetrahedral intermediate and the following transition state.

References[edit]

Berg, Jeremy "Biochemistry" Sixth edition. Freeman and Company, 2007