Understanding the Movement of Chemicals into and out of Enzyme Active Sites

Acetylcholinesterase (AChE) catalyzes the hydrolysis of the neurotransmitter acetylcholine into acetate ion and choline at nearly diffusion controlled rates. The position of the active site at the bottom of a 20 Å deep and narrow gorge raises questions regarding the existence of alternate channels for efficient product clearance. Preliminary molecular mechanics calculations using program YETI suggested that a channel ending at Arg244 is a possible alternate exit for acetic acid/acetate ion. Acetic acid/acetate ion exit were studied through the two competing pathways: the entrance gorge and the 6 Å shorter alternate exit channel. The potential of mean force (PMF) profiles for product exit through both pathways were calculated using the umbrella sampling technique combined with stochastic boundary molecular dynamics as implemented in CHARMM. The PMF for the migration of acetic acid decreases by ~ 8 kcal/mol after 8 Å travel through the main gorge, while the PMF for acetate ion exit falls by 14 kcal/mol. The interaction between acetic acid/acetate ion and the OH group of Tyr121 appears to guide product release through the main gorge. Acetic acid encounters a more than 6 kcal/mol barrier through the alternate pathway, while the PMF for acetate ion drops ~ 27 kcal/mol when it approaches Arg244. Full molecular dynamics simulations, free of restraint, result in the migration of acetate ion through the alternate channel but not through the main gorge.

Istvan Enyedy The Catholic University of America Chemistry Department/ Maloney Hall Washington DC 20064 email: istvan@bioorg.ee.cua.edu phone: 202-319-5349 or 5707 fax: 202-319-5381