Structural Biochemistry/Enzyme Catalytic Mechanism/Proteases/Chymotrypsin

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Overview[edit | edit source]

Chymotrypsin, a protease, is an enzyme that cleaves the carbonyl side of certain peptide bonds by both general acid-base catalysis, but primarily covalent catalysis. In this mechanism, a nucleophile becomes covalently attached to a substrate in a transition state with an acyl-enzyme. The protease cleaves proteins by a hydrolysis reaction, an addition of a water molecule. The double bond between the carbon and nitrogen strengthens its bond. Chymotrypsin is site specific and will only cleave the carboxyl side of large hydrophobic or aromatic amino acids such as phenylalanine (Phe), methionine (Met), tyrosine (Tyr), and tryptophan (Trp), unless the next amino acid is proline (Pro). The reason why chymotrypsin prefers to cleave specifically to bulky hydrophobic amino acids is due to the formation of S1 pockets,which, in the case of chymotrypsin, is lined with relatively hydrophobic residues such as Ser-189, Ser-214, Trp-215, Gly-216, and Gly-226. Chymotrypsin catalyzes the reaction rate by a factor of 109. The reaction has two steps, an acylation phase and a deacylation phase. In the former phase, the peptide bond is cleaved and an ester is formed between substrate and enzyme. In the latter phase, this ester is hydrolyzed and the enzyme is regenerated.

This illustrates the covalent catalysis of chymotrypsin. The first step is the acylation, which forms the acyl-enzyme intermediate. Then the acyl-enzyme intermediate goes through deacylation converting back to its original free enzyme form.

Evidence of Mechanism[edit | edit source]

Because chymotrypsin can also catalyze the hydrolysis of esters and amides, p-nitrophenolacetate was used in conjunction with chymotrypsin. The reaction with p-nitrophenolacetate will yield p-nitrophenol, a chromic-effector with a yellow color change in the product. The absorbency can be determined from the color and the intensity can determine the amount of product. Hartley and Kilby used this information in 1954 to show that the reaction proceeds in two phases: a Burst Phase and then levels off to a steady-state phase. Thus, there is a formation of a covalently bound enzyme substrate intermediate.

N-Acetyl-L-phenylalanine p-nitrophenyl ester yields a yellow product, p-Nitrophenolate, on cleavage by chymotrypsin.

Another test to determine the mechanism of chymotrypsin hydrolysis was to treat the protease with an organofluorophosphate, diisopropylphosphofluoridate (DIPF). In this reaction, chymotrypsin loses all activity and becomes inactivated. Since only serine-195 was modified by diisopropylphosphofluoridate, it indicates that Serine-195 plays the crucial role in the mechanism as a nucleophile. It is covalently linked to Serine-195. Covalent catalysis of chymotrypsin basically goes through acylation and deacylation. Acylation forms the acyl enzyme intermediate and the deacylation adds water which produces a free enzyme.

Site-directed mutagenesis is another technique that can test the reaction by creating a mutant in the amino acid sequence of the active site of the enzyme. It supported the mechanism below by demonstrating that the replacement through site directed mutagenesis of any one member of the catalytic triad had a devastating effect on reaction rate. In fact, replacing just one of the triad had the same effect as replacing all three--demonstrating that each component is vital for efficient catalysis. While the enzyme continued to bind to the substrate (we know this because the KM remained constant throughout the replacements--it required the same substrate concentration to achieve half of the maximum rate), the reaction rate was orders of magnitude smaller without the triad.

Structure of Chymotrypsin[edit | edit source]

The primary structure shows that disulfide bonds are the crucial role to the protein folding. The protein is spherical and itself consists of three polypeptide chains. There is also a pocket in the protein which is known as the active site. The active site includes Ser-195, His-57, and Asp-102 (the catalytic triad). Ser-195 is hydrogen bonded to the His-57 and it in turn is hydrogen bonded to the Asp-102 residue. The His-57 role is to position the serine residue and polarize the hydroxyl group so it can be deprotonated to the alkoxide ion. In the presence of the substrate, this accepts a proton by acting as a base. Asp-102 orients the His-57 and stabilizes it through hydrogen bonding and electrostatics.

Mechanism[edit | edit source]

Step 1: When substrate (polypeptide) binds, the side of chain of the residue next to the peptide bond to be cleaved nestles in a hydrophobic pocket on the enzyme, positioning the peptide bond for attack. Histidine extracts one proton from serine to form an alkoxide ion. This serine ion reacts with the substrate.

Step 2: In chymotrypsin, the carboxylate R-group of Asp102 forms a hydrogen bond with R group of His 57. When this happens, it compresses this hydrogen bond and shifts electron density to the other nitrogen atom (not involved in the H-bond) in the R-group of His57 becomes a very strong base. This allows His 57 to deprotonate Ser195 and turn it into a strong nucleophile that can attack the substrate.

Oxyanion hole stabilizes the tetrahedral intermediate. It is formed by hydrogen bonds linking peptide NH groups to the negatively charged oxygen atom.

Oxygen develops a partially negative charge in the oxyanion hole.

Step 3: Instability of the negative charge on the substrate carbonyl oxygen when will leads to collapse of the tetrahedral intermediate, re-formation of a double bond with carbon which breaks the peptide bond between the carbon and amino acid group. The amino leaving group is protonated by His57, facilitating its displacement. Once the oxyanion hole stabilizes the negative charge, the bond breaks because the proton from Histidine is binding to nitrogen to make it less likely to carbon. The leaving group is stabilized and the acyl-enzyme is formed.

Step 4: The amine component is departed from the enzyme (metabolized by the body) and binds to serine. This completes the first stage (acylation of enzyme). The first product has been made.

Step 5: A water molecule is added where the N terminus was. Histidine deprotonates the water to form a hydroxyl group. This hydroxyl group attaches to carbon from the carboxyl side and destabilizes the acyl intermediate. The bond is broken.

Step 6: An incoming water molecule is deprotonated by acid-base catalysis, generating a strongly nucleophilic hydroxide ion. Attack of hydroxide on the ester linkage of the acylenzyme generates a second tetrahedral intermediate.

Step 7: collapse of the tetrahedral intermediate form the second product, a carboxylate anion, and displace Ser195. The proton from Histidine goes back to Serine.

Step 8: The carboxylic acid is released and the enzyme is reformed to catalyze the next reaction with the original active site.