The Role of Amino‐Terminal Alanine in the Control of Conformation and Activity of α‐Chymotrypsin

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Abstract
Novel acetylated derivatives of three different three‐chained chymotrypsins were prepared from bovine chymotrypsinogen A and their catalytic properties and kinetics of denaturation in urea were compared with those of the corresponding non‐acetylated enzymes.Measurements of the Km (apparent) as a function of pH confirmed earlier findings of Valenzuela and Bender [J. Biol. Chem. 248, 4909–4914 (1973)] that the α‐species of chymotrypsin is much more sensitive to reversible inactivation at high pH compared to its sister three‐chained chymotrypsins. α1 and K‐chymotrypsin.Similarly, the denaturation rate constants in 8 M urea of α‐chymotrypsin were much more sensitive to high pH than α1 and K‐chymotrypsin. The urea denaturation study showed a transition at about pH 8 to a more urea‐sensitive from of α‐chymotrypsin, whereas α1 and K‐chymotrypsin were relatively insensitive to a change in pH from 6.5 to 10.When the N‐terminal Ala149 of α‐chymotrypsin was acetylated, thus preventing protonation of the N terminus, the active enzyme derivative displayed the same Km (app) vs pH profile as α1 and K‐chymotrypsin. Urea denaturation studies with this masked derivative also showed that the pH‐dependent transition of native α‐chymotrypsin at pH 8 was eliminated.These results demonstrate that it is the presence of the protonated Ala149 residue in α‐chymotrypsin that accounts for much of the hypersensitivity of this enzyme species to inactivation and urea denaturation in the pH region 7.5–10.