Recent x-ray structure for AChE places the active catalytic site deep within a gorge-like fold of the protein. Due to the topology of AChE, there exists an active site gorge of about 20A long, that penetrates basically halfway through the enzyme and widens near the base. Such a configuration of charge suggests an electrostatic mechanism for directing the positively charged ACh into the gorge and towards the active site. With such a mechanism, a fast turn-over rate is necessary for efficent catalysis of acetylcholine. A question arose as to the mechanism for eliminating the choline by-product. Bartolucci et al. wanted to see what would happen if they blocked the entrance of the gorge with a ligand, and then observed where the enzymatic product went; whether it remained within the gorge, or it exited through some previously unobserved location. The dimethylmorpholinooctylcarbamic moiety of MF268 was covalently bound to the catalytic serine 200, located at the bottom of the long and narrow gorge. The alkyl chain of the inhibitor fills the upper part of the gorge, blocking the entrance of the active site. This prevents eseroline, the leaving group of the carbamoylation process, from exiting through this path. After the reaction, it was noted that the relatively bulky eseroline was not found in the crystal structure. This brought into question the existence of an alternative route for its elimination. This represents indirect evidence that a "backdoor" opening may occur and shows that the release of products via a "backdoor" is a likely mechanism for this enzyme.

Huge Ligand (MF268) Interaction with Ser200

This view of AChE shows the ligand and Ser200.

Huge Ligand (MF268) Blocking Gorge

Shown here are Tyr70, Trp279 and the ligand MF268. Tyr70 and Trp279 mark the peripheral anionic site of the AChE molecule. It is quite evident the ligand is blocking the entrance and exit of other molecules at the gorge region. The research team was successful in binding a ligand into the active site gorge that would block its entrance.

Huge Ligand (MF268) Interaction with Narrow Junction

Shown here are MF268, Tyr121, and Phe330. Tyr121 and Phe330 form the most narrow portion of the active site gorge that separates the upper and lower portion of the AChE molecule.

Huge Ligand (MF268 )Full Picture

This model shows the ligand and its interacitons, as a whole, with all of the previously discussed residues it interacts with. As you will notice, there is no exit route for the catalytic product eseroline. The front of the gorge is blocked off, and there are no other apparent exits.

1st Hypothesized "backdoor"

Shown here are Trp84, Val129 in violet, and Gly441. A shutter-like motion is proposed to occur between these residues, therefore allowing the catalytic product to exit. Tyr70, shown as a wireframe, is a reference point toward the entrance of the active site gorge.

2nd Hypothesized "bac door"

This view shows the residues Cys67 and Cys94 with Tyr70 again shown a reference point toward the entrance of the active site gorge.. Bartolucci, et al. propose that a flap-like conformational transion could occur in the region between Cys67 and Cys94.

References

Bartolucci, C, Perola, E, Luciano, C, Mario, B, and Lamba, D. (1999) Biochemistry 38, 5714-5719.

Harel M, Schalk I, Ehret-Sabatier F, Goeldner M, Hirth C, Axelse H, Silman I, Sussman J. (1993)Proc.Natl.Acad.Sci. 90, 9031-9035.

Koellner G, Kryger G, Millard C, Silman I, Sussman, J, and Steiner T. (2000) 296, 713-735.

Ripoll D, Ferman C, Aselsen P, Silman I. (1993) Proc.Natl.Acad.Sci. 90, 5128-5132.

Sussman JL, Harel M, Silman I. (1993) Chem. Biol. Interact. 87, 187-97.

Szegletes T, Mallender W, and Rosenberry T. (1998) 37, 4026-4216 Szegletes T, Mallender W, Thomas P, Rosenberry T. (1999) Biochemistry 38, 122-133.