Kinetics of α-chymotrypsin action. I. pH, solvent, and temperature effects

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Abstract
An investigation has been made of the influence of pH on the kinetics of the [alpha]-chymotrypsincatalyzed hydrolysis of N-acetyl-L-tyrosine ethyl ester, p-nitrophenyl acetate, and N-benzoyl-D- and -L-alanine methyl esters. From the pH variations of [image]c, [image]m, and [image]c/[image]m, it is deduced that in the free enzyme there are ionizing groups of pK 6.9 and 9.2. From the variation of these pK values with dielectric constant, it is concluded that, when protonated, one group is cationic and the other neutral. A temperature-dependence study was carried out on N-benzoyl-D- and -L-alaine methyl esters. The sharp break in the plot of log [image]c against 1/T is attributed to a rapid reversible denaturation of the enzyme at the higher temperatures. In the low-temperature region, the activation energies are [DELTA]EL = 16.2[plus or minus] 0.3kcal/moleand [DELTA] ED = 16.5[plus or minus] 0.6 kcal/mole.
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