The effect of urea and guanidinium chloride on activity of subtilisin Carlsberg.

As shown by viscosity and optical rotation dispersion measurements, subtilisin Carlsberg is not denatured in the presence of 10 M urea or 6 M guanidinium chloride. This unusual structural stability made it possible to investigate the effects of these hydrophobic-bond breaking solutes on various aspects of the enzymic interaction with substrates and inhibitors. The binding of the competitive inhibitor N-benzoylarginine was decreased by urea or guanidinium chloride. The nature of this effect was such as to implicate hydrophobic interaction as making a major contribution to the binding. By contrast, Ks for the substrates N-acetyltyrosine ethyl ester, N-benzoylarginine ethyl ester and N-trans-cinnamoylimidazole was apparently unchanged by the presence of urea or guanidinium chloride. The influence of these solutes on kcat for the substrates was rather involved. Tentative hypotheses are put forward to account for the effects seen.