Chymotrypsin active-site titration

Such an intermediate is known to be formed in reactions catalyzed by trypsin, chymotrypsin, thrombin, other enzymes of the blood-clotting cascade (except angiotensinconverting enzyme, which is an aspartic protease). An acyl-serine intermediate is also formed in the acetylcholinesterase reaction. The active site serine of this enzyme and the serine proteases can be alkylated by diisopropyl-fluorophosphate. See also Active Site Titration... 

For a-chymotrypsin, the procedure of active-site titration for the calculation of active enzyme concentration and thus of the catalytic constant kcat is long established. The original active-site titration experiment on a-CT by Hartley and Kilby (Hartley, 1954) was performed with ethyl p-nitrobenzoate (Figure 9.2). 

Titration of the intact active site obviates problems due to inactive protein which contribute to a false molarity. Active-site titrations of acyl group transfer enzymes such as a-chymotrypsin utilise a substrate which has a good leaving group. This enables the buildup of an acyl enzyme intermediate which forms faster than it can degrade and results in... 

An exchangeable proton of ribonuclease A titrates with a pK of 5.8 and has been assigned [39] to the NH of the active-site histidine-119. A low field resonance can be observed for chymotrypsin in H2O [40] and its pH dependence (15 to 18 ppm, pKa 7.2) and response to chemical modification suggests that this is the hydrogen-bonded proton between His-57 and Asp-102 at the active site. 

N-(m/is-Cinnamoyllmidazole (1). Mol. wt. 198.22, m.p. 133-133.5°. Prepared in high yield by reaction of cinnamoyl chloride with imidazole in benzene at 10-25°. The reagent reacts rapidly and quantitatively with the active site of a-chymotrypsin and hence can be used for the spectrophotometric determination of the normality of an enzyme solution by titration. 

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