Screening of the active site from water by the incoming ligand triggers catalysis and inhibition in serine proteases

Abstract

The pKa of the catalytic His57 N(epsilon)H in the tetrahedral complex (TC) of chymotrypsin with trifluoromethyl ketone inhibitors is 4-5 units higher relative to the free enzyme (FE). Such stable TC's, formed with transition state (TS) analog inhibitors, are topologically similar to the catalytic TS. Thus, analysis of this pKa shift may shed light on the role of water solvation in the general base catalysis by histidine. We applied our QM/SCRF(VS) approach to study this shift. The method enables explicit quantum mechanical DFT calculations of large molecular clusters that simulate chemical reactions at the active site (AS) of water solvated enzymes. We derived an analytical expression for the pKa dependence on the degree of water exposure of the ionizable group, and on the total charge in the enzyme AS, Q(A) and Q(B), when the target ionizable functional group (His57 in this study) is in the acidic (A) and basic (B) forms, respectively. Q2(B) > Q2(A) both in the FE and in the TC of chymotrypsin. Therefore, water solvation decreases the relative stability of the protonated histidine in both. Ligand binding reduces the degree of water solvation of the imidazole ring, and consequently elevates the histidine pKa. Thus, the binding of the ligand plays a triggering role that switches on the cascade of catalytic reactions in serine proteases.

An Internal Water-Retention Site in the Rhomboid Intramembrane Protease GlpG Ensures Catalytic Efficiency

“…As such, our investigations have provided an intriguing and unexpected insight into the function of membrane-immersed proteases; guaranteeing water supply to the catalytic residues at the time of hydrolysis is indeed a deliberate strategy for ensuring catalysis proceeds efficiently. In fact, recent work suggests that catalysis by soluble serine proteases actually requires water desolvation from the active site (Shokhen et al, 2008). Water retention at a specialized site could thus even be more widely applicable to general proteases, especially those that process hydrophobic substrates, although this awaits further analysis.…”

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].…”

Application of EMBM to Structure‐Based Design of Warheads for Protease Inhibitors

“…[22] The heterogeneous combined protein/water environmental effect were accounted for by the QM/SCRF(VS) approach [23] by the continuum solvation COSMO algorithm [24] implemented in MOPAC2009. W1 and W2 were calculated for every CS on a wide interval (from 1 to 80) of dielectric constant of the polarized continuum.…”