The mechanism of chymotrypsin

Inside vivo, chymotrypsin is a proteolytic enzyme (serine protease) acting in the intestinal systems of many organisms. It facilitates the tits of peptide bonds by a hydrolysis reaction, which despite being thermodynamically favorable, occurs extremely slowly in the absence of a catalyst. The main substrates of chymotrypsin are peptide bonds in which the amino acid N-terminal to the bond is a tryptophan, tyrosine, phenylalanine, or leucine. Like many proteases, chymotrypsin also hydrolyzes amide bonds in vitro, a virtue that enabled the utilization of substrate analogs such as N-acetyl-L-phenylalanine p-nitrophenyl amide for enzyme assays.

System of peptide bond tits in α-chymotrypsin
Chymotrypsin cleaves peptide bonds by targeting the unreactive carbonyl group with a powerful nucleophile, the serine 195 deposits found in the active site of the enzyme, which briefly becomes covalently fused 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 results rely on inhibition assays and the study of the kinetics of tits of the aforementioned base, exploiting the point that the enzyme-substrate intermediate p-nitrophenolate has a yellow color, enabling measurement of its concentration by measuring light absorbance at 410 nm.

The response of chymotrypsin with its base was found to take place in two phases, an initial “burst” period at the beginning of the reaction and a steady-state phase following Michaelis-Menten kinetics. The mode of action of chymotrypsin clarifies this as hydrolysis requires a place in two steps. First, acylation of the substrate to form an acyl-enzyme intermediate, and then deacylation to return the enzyme to its original state. This particular occurs from your concerted action of the three-amino-acid elements in the catalytic triad. Aspartate hydrogen bonds to the N-δ hydrogen of histidine, increasing the pKa of its ε nitrogen, thus rendering it capable to deprotonate serine. This deprotonation allows the serine side cycle 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 surrounding main chain N-hydrogens. This occurs in the oxyanion hole. This forms a tetrahedral adduct and break of the peptide relationship. An acyl-enzyme intermediate, bound to the serine, is formed, and the freshly formed amino terminus of the cleaved protein can dissociate. In the second reaction step, a water molecule is activated by the essential histidine and works as a nucleophile. The oxygen of water attacks the carbonyl carbon of the serine-bound acyl party, resulting in the formation of any second tetrahedral adduct, reconstruction of the serine -OH group, and release of the proton, as well because of the protein fragment with the newly formed carboxyl joli