Editing Chymotrypsin (section)

== Mechanism of action and kinetics ==
{{missing information|which specific protein this residue numbering is based on (use hatnote)|date=June 2022}}
[[File:062821 Chymotrypsin Tetrahedral Intermediates.pdf|left|420px|alt=Chymotrypsin tetrahedral intermediates|thumb|Molecular mechanism of chymotrypsin-catalyzed hydrolysis of [[peptide bond]]. One key aspect is the tetrahedral intermediate Tet 1.]]
{{See also|Catalytic triad}}
''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 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 hydrolyses 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 [[4-Nitrophenol|''p''-nitrophenolate]] has a yellow colour, enabling  measurement of its concentration by measuring light absorbance at 410&nbsp;nm.

Chymotrypsin catalysis of the hydrolysis of a protein substrate (in red) is performed in two steps.  First, the nucleophilicity of Ser-195 is enhanced by general-base catalysis in which the proton of the serine hydroxyl group is transferred to the imidazole moiety of His-57 during its attack on the electron-deficient carbonyl carbon of the protein-substrate main chain (k1 step). This occurs via the concerted action of the three-amino-acid residues in the catalytic triad. The buildup of negative charge on the resultant tetrahedral intermediate is stabilized in the enzyme's active site's oxyanion hole, by formation of two hydrogen bonds to adjacent main-chain amide-hydrogens.

The His-57 imidazolium moiety formed in the k1 step is a general acid catalyst for the k-1 reaction.  However, evidence for similar general-acid catalysis of the k2 reaction (Tet2)<ref>{{Cite journal|last1=Fersht|first1=A.R.|last2=Requena|first2=Y.|date=1971|title=Mechanism of the -Chymotrypsin-Catalyzed Hydrolysis of Amides. pH Dependence of kc and Km.|journal=J. Am. Chem. Soc.|volume=93|issue=25|pages=7079–87|doi=10.1021/ja00754a066|pmid=5133099}}</ref> has been controverted;<ref>{{Cite journal|last1=Zeeberg|first1=B.|last2=Caswell|first2=M.|last3=Caplow|first3=M.|date=1973|title=Concerning a reported change in rate-determining step in chymotrypsin catalysis|journal=J. Am. Chem. Soc.|volume=95|issue=8|pages=2734–5|doi=10.1021/ja00789a081|pmid=4694533|bibcode=1973JAChS..95.2734Z }}</ref> apparently water provides a proton to the amine leaving group.

Breakdown of Tet1 (via k3) generates an acyl enzyme, which is hydrolyzed with His-57 acting as a general base  (kH2O) in formation of a tetrahedral intermediate, that breaks down to regenerate the serine hydroxyl moiety, as well as the protein fragment with the newly formed carboxyl terminus.