ON THE MECHANISM OF ACETYLCHOLINESTERASE ACTION - THE ELECTROSTATICALLY INDUCED ACCELERATION OF THE CATALYTIC ACYLATION STEP

Brownian dynamics simulations of the encounter kinetics between the ac tive site of the wild-type and Glu199 mutant of Torpedo californica ac etylcholinesterase (TcAChE) with a charged substrate were performed. I n addition, ab initio quantum chemical calculations using the 3-21G ba sis set were undertaken to probe the energetics of the transformation of the Michaelis complex into a covalently bound tetrahedral intermedi ate using various models of the wild-type and Glu199Gln mutant active sites. The quantum calculations predicted about a factor of 32 reducti on in the rate of formation of the tetrahedral intermediate upon the G lu199Gln mutation and showed that the Glu199 residue located in the pr oximity of the enzyme active triad boosts AChE's activity in a dual fa shion: (1) by increasing the encounter rate due to the favorable modif ication of the electric field inside the enzyme reaction gorge and (2) by stabilization of the transition state for the first chemical step of catalysis. Our calculations also demonstrate the critical role of t he oxyanion hole in stabilization of the tetrahedral intermediate and suggests that a charge relay mechanism may operate in the Glu199Gln mu tant AChE as opposed to a general base mechanism as in the wild-type e nzyme.