The role of amino-terminal alanine in the control of conformation and activity of alpha-chymotrypsin.
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 alpha-species of chymotrypsin is much more sensitive to reversible inactivation at high pH compared to its sister three-chained chymotrypsins, alpha 1 and kappa-chymotrypsin. Similarly, the denaturation rate constants in 8 M urea of alpha-chymotrypsin were much more sensitive to high pH than alpha 1 and kappa-chymotrypsin. The urea denaturation study showed a transition at about pH 8 to a more urea-sensitive form of alpha-chymotrypsin, whereas alpha 1 and kappa-chymotrypsin were relatively insensitive to a change in pH from 6.5 to 10. When the N-terminal Ala149 of alpha-chymotrypsin was acetylated, thus preventing protonation of the N terminus, the active enzyme derivative displayed the same Km (app) vs pH profile as alpha 1 and kappa-chymotrypsin. Urea denaturation studies with this masked derivative also showed that the pH-dependent transition of native alpha-chymotrypsin at pH 8 was eliminated. These results demonstrate that it is the presence of the protonated Ala149 residue in alpha-chymotrypsin that accounts for much of the hypersensitivity of this enzyme species to inactivation and urea denaturation in the pH region 7.5-10.
D000409:Alanine; D000595:Amino Acid Sequence; D002918:Chymotrypsin / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011487:Protein Conformation; D011489:Protein Denaturation
D002918:Chymotrypsin; D000409:Alanine