History of Chymotrypsin

In the early 1900s, Vernon proposed that pancreatic preparations could give rise to an intrinsic activator of its own enzymes (Vernon 1901). Vernon’s milk-clotting experiments determined there were at least two enzymes present and that one was more stable than the other (Vernon 1902). However, this idea was not widely accepted until 1934 when Kunitz and Northrop confirmed the presence of an enzyme in addition to trypsin, naming it chymotrypsin. They were able to crystallize chymotrypsin, as well as the inactive precursor, chymotrypsinogen (Kunitz and Northrop 1934). In 1938, Kunitz isolated different active forms of chymotrypsin, designating them as alpha, beta, and gamma (Kunitz 1938).

In the early 1940s Fruton and Bergmann further studied the specificity of chymotrypsin, reporting on several new substrates (Fruton and Bergmann 1942). Jacobsen soon identified additional forms of chymotrypsin, designating them as delta and pi (Jacobsen 1947). In 1948, Schwert further characterized the molecular weights of chymotrypsin and chymotrypsinogen.
In 1954, the first evidence for the three-step mechanism of chymotrypsin hydrolyzing amide and ester substrates was reported on by Hartley and Kilby, who hypothesized the presence of an acyl enzyme intermediate, which was later proven to be true (Henderson 1970).  In 1955, Laskowski obtained a second crystalline chymotrypsinogen, naming it chymotrypsinogen B. In 1964 Hartley determined the amino acid sequence of chymotrypsin A, which was later refined by Meloun et al. in 1966. In 1968, Smillie et al. determined the amino acid sequence of chymotrypsin B, which revealed 80% sequence identity with chymotrypsin A. Throughout the 1970s and 1980s research was done to better understand the mechanism of action, and identify the differences in amino acid sequences between trypsin and chymotrypsin (Steitz et al. 1969, Cohen et al. 1981, Asbóth and Polgár 1983, and Gráf et al. 1988).
In the 1990s, chymotrypsin was purified from other sources including Atlantic cod (Ásgeirsson and Bjarnason 1991), and camel (Al-Ajlan and Bailey 1997). Work also begun on investigating inhibitors (Baek et al. 1990), and Frigerio et al. elucidated the crystal structure of bovine chymotrypsin to a 2.0 Å resolution.
Recent research has investigated the folding and denaturation of chymotrypsin over a range of concentrations (Ghaouar et al. 2010), chymotrypsin’s interaction with nanoparticle substrates (You et al. 2006, and Jordan et al. 2009), and increasing chymotrypsin stability by conjugating to PEG molecules.

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