The question of differential hydrogen bonding in the mechanism of catalysis by serine proteases

Two mechanisms recently proposed to account for the action of serine in proteases differ on the matter of hydrogen bonding between enzyme and substrate in the Michaelis complex and acyl enzyme. One mechanism involves the removal of hydrogen bonds as a means of raising the free energy of the Michaelis complex toward that of the transition state and thereby effecting catalysis. This report shows that the kinetic parameters $\text{k}{}_2\text{K}{}_{\mathrm{<mtext>s</mtext>}}$ and k₃ for the α-chymotrypsin-catalyzed hydrolysis of a series of halogen-substituted phenyl acetates respond to electron with-drawal and concomitant changes in hydrogen bond energy in a nearly identical fashion. Reference to thermodynamic studies of hydrogen bonding in free solution shows that these data are not consistent with hydrogen bond removal in the Michaelis complex and acyl enzyme but do support the alternative mechanism, in which hydrogen bonding of substrate is maintained along the entire reaction coordinate.