Chymotrypsin

CAS No:	9004-07-03
EINECS No:	232-671-2
EC No:	3.4.21.1
Synonyms:	α-chymotrypsin, chymotrypsin A, pancreatic chymotrypsin

Product Summary

Chymotrypsin is a powerful and widely used serine protease with highly selective cleavage activity toward peptide bonds of aromatic amino acids. Its roles in biomedical therapy, protein characterization, and biotech manufacturing make it a vital tool in both research and industry.

Function

Chymotrypsin catalyzes the hydrolysis of peptide bonds, especially those adjacent to aromatic amino acids such as phenylalanine, tyrosine, and tryptophan. It is naturally produced in pancreas as inactive zymogen chymotrypsinogen, which is activated in digestive tract.

Catalytic triad: Chymotrypsin contains a Ser₁₉₅–His₅₇–Asp₁₀₂ triad in its active site. This trio facilitates nucleophilic attack on the peptide bond carbonyl carbon, leading to bond cleavage.
Substrate specificity: It prefers peptide bonds where the carboxyl group is contributed by an aromatic amino acid due to a hydrophobic binding pocket.
Two-step reaction:
- Acylation: Formation of a covalent acyl-enzyme intermediate.
- Deacylation: Water hydrolyzes the intermediate, releasing the cleaved peptide.
- Optimal condition: pH 7.0-8.0, temperature: 37-45°C.
- Inhibitors: PMSF, aprotinin.

Applications in Scientific Research

Proteomics: Used to cleave proteins at aromatic amino acids in protein sequencing and digestion prior to mass spectrometry (MS).
Enzyme kinetics: A model enzyme for studying catalytic mechanisms and structure–function relationships.
Protein folding studies: Acts as a protease control to test refolding of proteins.
Tissue dissociation: In combination with trypsin or collagenase for cell isolation in cell culture.

Packaging & Storage

Available as white lyophilized powder.
Store in an airtight container at a temperature of 2 to 8°C, protected from light.

References

Bender ML, et al. 1964: The anatomy of an enzymatic catalysis. α-chymotrypsin, J Am Chem Soc. 86(18):3714–21.
Wells GB, et al. 1994: Structure at the active site of an acylenzyme of alpha-chymotrypsin and implications for the catalytic mechanism. An electron nuclear double resonance study, J Biol Chem. 269(6):4577-86.
Appel W. 1986: Chymotrypsin: Molecular and catalytic properties, Clin Biochem. 19(6):317-22.
Robillard G, Shulman RG. 1974: High resolution nuclear magnetic resonance studies of the active site of chymotrypsin. I. The hydrogen bonded protons of the "charge relay" system, J Mol Biol. 86(3):519-40.
Robillard G & Shulman RG. et al. 1974: High resolution nuclear magnetic resonance studies of the active site of chymotrypsin: II. Polarization of histidine 57 by substrate analogues and competitive inhibitors, J Mol Biol. 86(3):541-58.
Cruickshank WH, Kaplan H. 1975: Properties of the histidine residues of indole-chymotrypsin. Implications for the activation process and catalytic mechanism, Biochem J. 147(3):411–6.
Petrillo T, et al. 2012: Importance of tetrahedral intermediate formation in the catalytic mechanism of the serine proteases chymotrypsin and subtilisin, Biochem. 51(31):6164-70.
Giansanti P, et al. 2016: Six alternative proteases for mass spectrometry-based proteomics beyond trypsin, Nat Protoc. 11(5):993-1006.
Ma W, et al. 2005: Specificity of trypsin and chymotrypsin: loop motion controlled dynamic correlation as a determinant, Biophys J. 89(2):1183–93.
Freer ST, et al. 1970: Chymotrypsinogen: 2.5-angstrom crystal structure, comparison with alpha-chymotrypsin, and implications for zymogen activation, Biochem. 9(9):1997-2009.
Wang S, et al. 2009: Chymotryptic proteolysis accelerated by alternating current for MALDI-TOF-MS peptide mapping, J Proteom. 72(4):640-7.
Lathia US, et al. 2009: Development of inductively coupled plasma-mass spectrometry (ICP-MS) based protease assays, Anal Biochem. 398(1):93–98.