Chymotrypsin - Action and Kinetics of Chymotrypsin

Action and Kinetics of Chymotrypsin

In vivo, chymotrypsin is a proteolytic enzyme (Serine protease) acting in the digestive systems of many organisms. It facilitates the cleavage of peptide bonds by a hydrolysis reaction, which despite being thermodynamically favourable occurs extremely slowly in the absence of a catalyst. The main substrates of chymotrypsin include tryptophan, tyrosine, phenylalanine, leucine, and methionine, which are cleaved at the carboxyl terminal. Like many proteases, chymotrypsin will also hydrolyse amide bonds in vitro, a virtue that enabled the use of substrate analogs such as N-acetyl-L-phenylalanine p-nitrophenyl amide for enzyme assays.

Chymotrypsin cleaves peptide bonds by attacking the unreactive carbonyl group with a powerful nucleophile, the serine 195 residue located in the active site of the enzyme, which briefly becomes covalently bonded to the substrate, forming an enzyme-substrate intermediate. Along with histidine 57 and aspartic acid 102, this serine residue constitutes the catalytic triad of the active site.

These findings rely on inhibition assays and the study of the kinetics of cleavage of the aforementioned substrate, exploiting the fact that the enzyme-substrate intermediate p-nitrophenolate has a yellow colour, enabling us to measure its concentration by measuring light absorbance at 410 nm.

It was found that the reaction of chymotrypsin with its substrate takes place in two stages, an initial “burst” phase at the beginning of the reaction and a steady-state phase following Michaelis-Menten kinetics. It is also called "ping-pong" mechanism. The mode of action of chymotrypsin explains this as hydrolysis takes place in two steps. First acylation of the substrate to form an acyl-enzyme intermediate and then deacylation in order to return the enzyme to its original state. This occurs via the concerted action of the three amino acid residues in the catalytic triad. Aspartate hydrogen bonds to the N-δ hydrogen of histidine, increasing the pKa of its ε nitrogen and thus making it able to deprotonate serine. It is this deprotonation that allows the serine side chain to act as a nucleophile and bind to the electron-deficient carbonyl carbon of the protein main chain. Ionization of the carbonyl oxygen is stabilized by formation of two hydrogen bonds to adjacent main chain N-hydrogens. This occurs in the oxyanion hole. This forms a tetrahedral adduct and breakage of the peptide bond. An acyl-enzyme intermediate, bound to the serine, is formed, and the newly formed amino terminus of the cleaved protein can dissociate. In the second reaction step, a water molecule is activated by the basic histidine, and acts as a nucleophile. The oxygen of water attacks the carbonyl carbon of the serine-bound acyl group, resulting in formation of a second tetrahedral adduct, regeneration of the serine -OH group, and release of a proton, as well as the protein fragment with the newly formed carboxyl terminus