TIBS 1 6 -
OCTOBER1991
JOURNALCLUB Acetvlcholine
mustard did not label the binding site Acetylcholine esterase (ACHE, acetyl(G. Weiss and A. Maelicke, submitted) choline hydrolase, EC 3.1.1.7) plays a but aromatic residues were labeled by key role in cholinergic neurotransmis[3H]DDF ( p-N,N-dimethylaminobenzenesion. By rapid hydrolysis of the transdiazonium fluoroboratey s.'6, suggesting mitter acetylcholine (ACh), the enzyme that ionized aromatic ring systems effectively terminates the chemical rather than carboxylic groups act as impulse, thereby setting the basis for counter structures. rapid, repetitive responses and Although an AChE was first crystalenabling the re-uptake (and recycling) of choline. The enzyme was first of serine hydrolases, with a histidine lized in 1967 (Ref. 17) the packing of the described by David Nachmansohn in implicated as the intermediary charge crystals obtained was not ideal for structural analysis. Crystals with a 1938. In the following four decades, relay residue m. There is an ongoing dispute as to the more suitable space group have now Nachmansohn and his school solved the central features of the enzyme's structure of the acceptor site for the been obtained Is and the X-ray analysis mechanism of action, and provided the trimethylammonium head group of ACh of these by Sussman, Silman and colbasis for an understanding, at the in the three major macromolecules that leaguess provides a solid basis for an inmolecular level, of the action of acetyl- interact with ACh, i.e. acetylcholine depth molecular analysis of the struccholine esterase inhibitors as drugs and esterase, and the nicotinic and mus- tural properties of this important poisons ~.~. More recently, it became carinic acetylcholine receptors (n~ChR, enzyme. The structure of AChE (Fig. I) is strikclear that the enzyme exists in various mAChR). For ACHE, Cohen and cool~gomeric forms, which are membrane workers" have suggested that the ingly similar to the recently reported anchored or freely diffusible3, and cDNA 'anionic' sub-site is uncharged and structures of several other serine cloning and sequencing has revealed lipophilic, on the basis of studies hydrolases tg-~, and to a lipase23 and a the primary structures of several employing cationic and uncharged haloalkane dehalogenase z4. In agreeAChEs4. Sussman, Silman and col- homologs of ACh. Studies by Silman ment with recent findings4,zs, the cataleagues have now solved the three- and colleagues ~a~3 and Goeldner and lytic triad contains a serine and a histidimensional atomic structure of AChEs. Hirth ~4 suggested the presence of aro- dine residue (Ser200 and His440). In Certain features of the structure of the matic residues in the ACh binding contrast to the usual Asp residue, active site have important implications pocket. For the nAChR, an acetylcholine however, the third residue of the triad for the interaction of acetylcholine with acetylcholine receptors. Acetylchollne esterase is a serine hydrolase, reacting with its natural substrate at i close to diffusion-controlled rate ~. The trimethylammonium head group of acetylcholine binds to the 'anionic' sub-site of the enzyme's active site and, the ester-bond region to the 'esteratic' sub-site9. A key step in the reaction mechanism is the acylation of a serine residue in the enzyme's active site (Ser200 in the Torpedo califomica enzyme4); the acute toxicity of carbamoyl esters and organic fluorophosphates is caused by Rgum 1 the very slow hydrolysis Stereo ribbon diagram of the AChE monomer. The amino terminus is located at the top right of the rates of such acyl-enzyme picture, and the carboxyl terminus at the lower left The 12-stranded ~sheet is approximately parallel to the plane of the figure, with its convex surface pointing up. The sheet is sandwiched between six intermediates. The active helices, two on its concave surface (underneath in this view) and four on its convex surface. The other site is believed to contain eight helices all occur in loops above the sandwich. the catalytical triad typical 355 © 1991,ElsevierSciencePublishers,(UK) 0376-5067/911502.00
este ase: the stFuctu e
'
,,
i
i
TIBS 1 6 - OCTOBER 1 9 9 1
is a Glu (Glu327), just as in the lipase trom Geotrichum candidum ~. in addition, the triad displays opposite handedness to that of prototype serine enzymes, such as chymotrypsin. The most exciting feature of the structure is a d~.ep and narrow gorge, about 20 ~. long, which penetrates halfway into the enzyme and widens close to its base (Fig. 2). This gorge contains the catalytic triad and thus must form the binding pocket for ACh. Most interestRgum 2 ingly, 14 aromatic Stereo drawing of a thin slice through the AChE monomer. The blue-dot van der Waals surface shows the residues line a narrow gorge leading down to the active site. The backbone is pink, the aromatic groups are light green and substantial portion the catalytic triad is white. (-40%) of the surface of the gorge. These residues and their flanking sequences choline from the active site. The almost 17 Leuzinger,W. and Baker,A. L. (1967) Proc. Natl Acad. ScL USA 57, 446-451 are highly conserved in AChEs from dif- identical association kinetics of ACh 18 Sussman,J. L. et aL (1989) in Proc. 7th Annu. ferent species 4.z~. In contrast, the gorge binding to AChE and nAChR29 suggests Chem. Defense BioscL Rev. pp. 309-316 contains only a few acidic residues, that other ACh-binding proteins have 19 Pathak, D. and Ollis, D. (1990) J. Mol. Biol. 214, 497-525 which are either hydrogen-bonded similar structures. Certainly, the X-ray (Asp72), or located at the top of the structure of AChE provides plenty of 20 Liao, D. and Remington,S. J. (1990) J. Biol. Chem. 265, 6528-6531 gorge (Asp285, Glu273), with the excep- food for further thoughts and studies. 21 Winkler, F. K., D'Arcy,A. and Hunziker,W. tion of Glu199 which is found at the bot(1990) Nature 343, 771-774 22 Brady, L. et aL (1990) Nature 343, 767-770 tom. If ACh is fitted manually into the References 23 Schrag,J. D. et aL (1991) Nature 351, gorge, the Glu199 residue makes close 1 Nachmanscinn,D. (1955) Harvey Lectures 761-764 1953/1954, pp. 57-99, AcademicPress contacts with one of the methyl groups 24 Franken, S. M. et at, (1991) EMBO J. 10, 2 Nachmansohn, D. and Neumann, E. (1975) of the trimethylammenium head and an 1297-1302 Chemic-31and Molecular Basis of Nerve Activity, 25 Gibney,G. et aL (1990) Proc. Natl Acad. ScL e~-carbon of the choline moiety. Since Academic Press USA 87, 7546-7550 Glu199 appears hydrogen-bonded to 3 Massouli(~,J. and Toutant, J-P.(1988) in 26 Gibney,G. et aL (1988) J. BioL Chem. 263, Handbook of Experimental Pharmacology (The Glu443 and its replacement by glu1140-1145 Cholinergic Synapse, Vol. 86) (Whittaker, V. P., 27 Berman, H. A. and Decker, M. M. (1989) J. BioL tamine does not significantly change ed.), pp. 167-224, Springer-Verlag Chem. 264, 3942-3950 the enzyme kinetics 25, it probably does 4 MacPhee-Quigley,K., Taylor,P. and Taylor,S. 28 Rosenberry,T. L. and Neumann,E. (1977) not form the major electronegative (1985) J. Biol. Chem. 260, 12185-12189 Biochemistry 16, 3870-3878 acceptor group. It is therefore likely 5 Sussman,J. L. et aL Science (in press) 29JOrss, R., Prinz, H. and Maelicke,A. (1979) 6 Hillman, G. R. and Mautner, H. G. (1970) Prec. Natl Acad. ScL USA 76, 1064-1068 that, as already suggested for the Biochemistry 9, 1749-1754 nAChR, ionized rings of aromatic amino 7 J~rss, R. and Maelicke,A. (1981) J. BioL Chem. 266, 2887-2893 ALFRED MAELICKE acid residues represent major elements 8 Bazelyansky,M., Robey,C. and Kirsch, J. F. of the 'anionic' site of ACHE. (1986) b'iochemistry25, 125-130 Institut for Physiologische Chemie und The high number of aromatic 9 Rosenberry,T. L. (1975) Adv. Enzymol. 43, Pathobi~chemie, Universit~t Mainz, residues of the active site gorge may 103-218 Duesbergweg 6, W-6500 Mainz, FRG. also be related to the existence of vari- 10 Wilson, I. B. and Bergmann,F. (1950) J. Biol. Chem. 186, 683-692 ous 'peripheral' binding sites for ACh 11 Cohen, S. G. et aL (1989) Biochim. Biophys. indicated by biochemical studies 9,11,27. Acta 997, 167-175 Don't miss The aromatic lining of the gorge may 12 Blumberg,S. and Silman,I. (1978) Biochemistry 17, 1125-1130 allow a mechanism of ACh interaction Substrate-binding sites in consisting of initial adsorption at a 13 Shinitzky,M., Dudai,Y. and SUman, I. (1973) acetylcholinesterases, FEBS Lett. 30, 125-128 'peripheral' site followed by two-dimen- 14 Goeldner, M. P. and Hirth, C. G. (1980) Proc. a review by F. Hucho Natl Acad. Sci. USA 77, 6439-6442 sional diffusion to the active site 28. Such 15 Dennis, M. et a!. (1988) Biochemistry 27, in the November issue of an aromatic guidance mechanism may 2346-2357 explain the high on-rates of substrate 16 Galzi, J-L. et al. (1990) J. BioL Chem. 265, TIPS binding and the rapid clearance of 10430-10437 356
OCTOBER1991
JOURNALCLUB Acetvlcholine
mustard did not label the binding site Acetylcholine esterase (ACHE, acetyl(G. Weiss and A. Maelicke, submitted) choline hydrolase, EC 3.1.1.7) plays a but aromatic residues were labeled by key role in cholinergic neurotransmis[3H]DDF ( p-N,N-dimethylaminobenzenesion. By rapid hydrolysis of the transdiazonium fluoroboratey s.'6, suggesting mitter acetylcholine (ACh), the enzyme that ionized aromatic ring systems effectively terminates the chemical rather than carboxylic groups act as impulse, thereby setting the basis for counter structures. rapid, repetitive responses and Although an AChE was first crystalenabling the re-uptake (and recycling) of choline. The enzyme was first of serine hydrolases, with a histidine lized in 1967 (Ref. 17) the packing of the described by David Nachmansohn in implicated as the intermediary charge crystals obtained was not ideal for structural analysis. Crystals with a 1938. In the following four decades, relay residue m. There is an ongoing dispute as to the more suitable space group have now Nachmansohn and his school solved the central features of the enzyme's structure of the acceptor site for the been obtained Is and the X-ray analysis mechanism of action, and provided the trimethylammonium head group of ACh of these by Sussman, Silman and colbasis for an understanding, at the in the three major macromolecules that leaguess provides a solid basis for an inmolecular level, of the action of acetyl- interact with ACh, i.e. acetylcholine depth molecular analysis of the struccholine esterase inhibitors as drugs and esterase, and the nicotinic and mus- tural properties of this important poisons ~.~. More recently, it became carinic acetylcholine receptors (n~ChR, enzyme. The structure of AChE (Fig. I) is strikclear that the enzyme exists in various mAChR). For ACHE, Cohen and cool~gomeric forms, which are membrane workers" have suggested that the ingly similar to the recently reported anchored or freely diffusible3, and cDNA 'anionic' sub-site is uncharged and structures of several other serine cloning and sequencing has revealed lipophilic, on the basis of studies hydrolases tg-~, and to a lipase23 and a the primary structures of several employing cationic and uncharged haloalkane dehalogenase z4. In agreeAChEs4. Sussman, Silman and col- homologs of ACh. Studies by Silman ment with recent findings4,zs, the cataleagues have now solved the three- and colleagues ~a~3 and Goeldner and lytic triad contains a serine and a histidimensional atomic structure of AChEs. Hirth ~4 suggested the presence of aro- dine residue (Ser200 and His440). In Certain features of the structure of the matic residues in the ACh binding contrast to the usual Asp residue, active site have important implications pocket. For the nAChR, an acetylcholine however, the third residue of the triad for the interaction of acetylcholine with acetylcholine receptors. Acetylchollne esterase is a serine hydrolase, reacting with its natural substrate at i close to diffusion-controlled rate ~. The trimethylammonium head group of acetylcholine binds to the 'anionic' sub-site of the enzyme's active site and, the ester-bond region to the 'esteratic' sub-site9. A key step in the reaction mechanism is the acylation of a serine residue in the enzyme's active site (Ser200 in the Torpedo califomica enzyme4); the acute toxicity of carbamoyl esters and organic fluorophosphates is caused by Rgum 1 the very slow hydrolysis Stereo ribbon diagram of the AChE monomer. The amino terminus is located at the top right of the rates of such acyl-enzyme picture, and the carboxyl terminus at the lower left The 12-stranded ~sheet is approximately parallel to the plane of the figure, with its convex surface pointing up. The sheet is sandwiched between six intermediates. The active helices, two on its concave surface (underneath in this view) and four on its convex surface. The other site is believed to contain eight helices all occur in loops above the sandwich. the catalytical triad typical 355 © 1991,ElsevierSciencePublishers,(UK) 0376-5067/911502.00
este ase: the stFuctu e
'
,,
i
i
TIBS 1 6 - OCTOBER 1 9 9 1
is a Glu (Glu327), just as in the lipase trom Geotrichum candidum ~. in addition, the triad displays opposite handedness to that of prototype serine enzymes, such as chymotrypsin. The most exciting feature of the structure is a d~.ep and narrow gorge, about 20 ~. long, which penetrates halfway into the enzyme and widens close to its base (Fig. 2). This gorge contains the catalytic triad and thus must form the binding pocket for ACh. Most interestRgum 2 ingly, 14 aromatic Stereo drawing of a thin slice through the AChE monomer. The blue-dot van der Waals surface shows the residues line a narrow gorge leading down to the active site. The backbone is pink, the aromatic groups are light green and substantial portion the catalytic triad is white. (-40%) of the surface of the gorge. These residues and their flanking sequences choline from the active site. The almost 17 Leuzinger,W. and Baker,A. L. (1967) Proc. Natl Acad. ScL USA 57, 446-451 are highly conserved in AChEs from dif- identical association kinetics of ACh 18 Sussman,J. L. et aL (1989) in Proc. 7th Annu. ferent species 4.z~. In contrast, the gorge binding to AChE and nAChR29 suggests Chem. Defense BioscL Rev. pp. 309-316 contains only a few acidic residues, that other ACh-binding proteins have 19 Pathak, D. and Ollis, D. (1990) J. Mol. Biol. 214, 497-525 which are either hydrogen-bonded similar structures. Certainly, the X-ray (Asp72), or located at the top of the structure of AChE provides plenty of 20 Liao, D. and Remington,S. J. (1990) J. Biol. Chem. 265, 6528-6531 gorge (Asp285, Glu273), with the excep- food for further thoughts and studies. 21 Winkler, F. K., D'Arcy,A. and Hunziker,W. tion of Glu199 which is found at the bot(1990) Nature 343, 771-774 22 Brady, L. et aL (1990) Nature 343, 767-770 tom. If ACh is fitted manually into the References 23 Schrag,J. D. et aL (1991) Nature 351, gorge, the Glu199 residue makes close 1 Nachmanscinn,D. (1955) Harvey Lectures 761-764 1953/1954, pp. 57-99, AcademicPress contacts with one of the methyl groups 24 Franken, S. M. et at, (1991) EMBO J. 10, 2 Nachmansohn, D. and Neumann, E. (1975) of the trimethylammenium head and an 1297-1302 Chemic-31and Molecular Basis of Nerve Activity, 25 Gibney,G. et aL (1990) Proc. Natl Acad. ScL e~-carbon of the choline moiety. Since Academic Press USA 87, 7546-7550 Glu199 appears hydrogen-bonded to 3 Massouli(~,J. and Toutant, J-P.(1988) in 26 Gibney,G. et aL (1988) J. BioL Chem. 263, Handbook of Experimental Pharmacology (The Glu443 and its replacement by glu1140-1145 Cholinergic Synapse, Vol. 86) (Whittaker, V. P., 27 Berman, H. A. and Decker, M. M. (1989) J. BioL tamine does not significantly change ed.), pp. 167-224, Springer-Verlag Chem. 264, 3942-3950 the enzyme kinetics 25, it probably does 4 MacPhee-Quigley,K., Taylor,P. and Taylor,S. 28 Rosenberry,T. L. and Neumann,E. (1977) not form the major electronegative (1985) J. Biol. Chem. 260, 12185-12189 Biochemistry 16, 3870-3878 acceptor group. It is therefore likely 5 Sussman,J. L. et aL Science (in press) 29JOrss, R., Prinz, H. and Maelicke,A. (1979) 6 Hillman, G. R. and Mautner, H. G. (1970) Prec. Natl Acad. ScL USA 76, 1064-1068 that, as already suggested for the Biochemistry 9, 1749-1754 nAChR, ionized rings of aromatic amino 7 J~rss, R. and Maelicke,A. (1981) J. BioL Chem. 266, 2887-2893 ALFRED MAELICKE acid residues represent major elements 8 Bazelyansky,M., Robey,C. and Kirsch, J. F. of the 'anionic' site of ACHE. (1986) b'iochemistry25, 125-130 Institut for Physiologische Chemie und The high number of aromatic 9 Rosenberry,T. L. (1975) Adv. Enzymol. 43, Pathobi~chemie, Universit~t Mainz, residues of the active site gorge may 103-218 Duesbergweg 6, W-6500 Mainz, FRG. also be related to the existence of vari- 10 Wilson, I. B. and Bergmann,F. (1950) J. Biol. Chem. 186, 683-692 ous 'peripheral' binding sites for ACh 11 Cohen, S. G. et aL (1989) Biochim. Biophys. indicated by biochemical studies 9,11,27. Acta 997, 167-175 Don't miss The aromatic lining of the gorge may 12 Blumberg,S. and Silman,I. (1978) Biochemistry 17, 1125-1130 allow a mechanism of ACh interaction Substrate-binding sites in consisting of initial adsorption at a 13 Shinitzky,M., Dudai,Y. and SUman, I. (1973) acetylcholinesterases, FEBS Lett. 30, 125-128 'peripheral' site followed by two-dimen- 14 Goeldner, M. P. and Hirth, C. G. (1980) Proc. a review by F. Hucho Natl Acad. Sci. USA 77, 6439-6442 sional diffusion to the active site 28. Such 15 Dennis, M. et a!. (1988) Biochemistry 27, in the November issue of an aromatic guidance mechanism may 2346-2357 explain the high on-rates of substrate 16 Galzi, J-L. et al. (1990) J. BioL Chem. 265, TIPS binding and the rapid clearance of 10430-10437 356
