A mixed mechanistic-electrostatic model to explain pH dependence of glycosyl hydrolase enzyme activity

Glycosyl hydrolases are a vast group of enzymes that share a common topology at their active site with two acidic residues that are responsible for activity. In spite of their similarity, they exhibit a wide range of pH optima that must depend on other factors. Using structural and mechanistic knowledge about glycosyl hydrolases from families 7, 10, and 16, we have formulated a new mathematical model that can include not only the ionization behavior of the catalytic residues but also as many ionizable residues as desired in the active site. In addition, the model can incorporate electrostatic influences via acid dissociation equilibrium constants and chemical relationships such as hydrogen bonds. The results of the simulations indicate a clear shift in the pH dependence of activity for the enzymes only when a close interrelation (hydrogen bond) between the catalytic and auxiliary residues in the active site is taken into account. This explains the observations from mutagenesis studies that show this type of shift and cannot be explained by a purely electrostatic interaction theory. Moreover, the presence of the kind of chemical interaction proposed could provide stabilization of the activity in the presence of environmental, structural, pH and electrostatic variations. These findings and the implications for the design of new mutagenesis strategies are discussed. The results suggest a way to modify, via site-directed mutagenesis, the acid dissociation of one of the catalytic residues in the active site independently of the other, which could have clear advantages over the purely electrostatic modifications that usually affect both residues simultaneously.