Factors Determining the Stability of Anionic Tetrahedral Complexes in Serine Protease Catalysis and Inhibition

Quantum mechanical ab initio (RHF/6-31+G*//RHF/3-21G) calculations were used to simulate the formation of the tetrahedral complex intermediate (TC) in serine protease active site by substrates and transition-state analog inhibitors. The enzyme active site was simulated by an assembly of the amino acids participating in catalysis, whereas the substrates and inhibitors were simulated by small ligands, acetamide (1) and trifluoroacetone (2), respectively. For the first time, the principal factors determining the relative stability of the TC in serine proteases are arranged according to their energy contributions. These include (a) formation of the new covalent bond between Ser195 Oγ and the electrophilic center of a ligand; (b) stabilization of the oxyanion in the oxyanion hole; (c) basic catalysis by His57; and (d) hydrogen bond between Asp102 carboxylate and Nδ of the protonated His57. We have directly calculated the gas-phase relative free energy of formation of TCAS(2) and TCAS(1), the value of ΔΔGg[TCAS(2,1)]. It is ΔEcov, the relative energy of the new covalent bond between the enzyme and the ligand formed in a TC that determines the experimentally observed large difference in the stability of TCs formed by substrates and TS-analog inhibitors of serine proteases. We demonstrated that the relative stability of TCs formed by a series of mono- and dipeptide amides and TFKs, derived from experimental kinetic data, can be rather well approximated by the sum of the theoretically calculated value of ΔΔGg[TCAS(2,1)] and the difference in hydration free energies of isolated ligands. Proteins 2000;40:154–167. © 2000 Wiley-Liss, Inc.