How do serine proteases really work?

Abstract

Recent advances in genetic engineering have led to a growing acceptance of the fact that enzymes work like other catalysts by reducing the activation barriers of the corresponding reactions. However, the key question about the action of enzymes is not related to the fact that they stabilize transition states but to the question to how they accomplish this task. This work considers the catalytic reaction of serine proteases and demonstrates how one can use a combination of calculations and experimental information to elucidate the key contributions to the catalytic free energy. Recent reports about genetic modifications of the buried aspartic group in serine proteases, which established the large effect of this group (but could not determine its origin), are analyzed. Two independent methods indicate that the buried aspartic group in serine proteases stabilizes the transition state by electrostatic interactions rather than by alternative mechanisms. Simple free energy considerations are used to eliminate the double proton-transfer mechanism (which is depicted in many textbooks as the key catalytic factor in serine proteases). The electrostatic stabilization of the oxyanion side of the transition state is also considered. It is argued that serine proteases and other enzymes work by providing electrostatic complementarity to the changes in charge distribution occurring during the reactions they catalyze.

Oxyanion and tetrahedral intermediate stabilisation by subtilisin: Detection of a new tetrahedral adduct

“…It has been suggested that the binding of substrates or structurally related inhibitors will induce a conformational change resulting in steric compression between histidine-57 and aspartate-102 and the formation of a low barrier hydrogen bond raising the pK a of histidine-57 [37,38]. Recent calculations [39,40] support the proposal [15,41] that inhibitor or substrate binding causes desolvation of the active site histidine residue in the serine proteases raising the pK a of the histidine residue and allowing it to be an effective general base catalyst enhancing the nucleophilicity of the hydroxyl group of serine-195 [15,21,33,41]. Raising the histidine pKa also allows it to act as a general acid catalyst for the breakdown of the tetrahedral intermediate [13-15, 21, 42] and to stabilize oxyanion formation [15,21].…”

Complement factor D, a novel serine protease

“…of chymotrypsin-like proteases is not accessible to the solvent and forms an H-bond with Asplo2, thus making an important contribution to its topology ( Fig. 5 ; Warshel et al, 1989). In both molecules of factor D, the hydroxyl group of Thr2I4 is pointed away from Asp'", failing to affect its orientation (Fig.…”

Regulation of serine protease activity by an engineered metal switch

“…petent configuration in the active site (Warshel et al, 1989). Likewise, it is possible that a bound metal cation in the vicinity of His-57 could destabilize the positive charge developed on this amino acid residue in the transition state.…”