title: N-acetyl-beta-D-glucopyranosylamine: a potent T-state inhibitor of glycogen phosphorylase. A comparison with alpha-D-glucose.
abstract: Structure-based drug design has led to the discovery of a number of glucose analogue inhibitors of glycogen phosphorylase that have an increased affinity compared to alpha-D-glucose (Ki = 1.7 mM). The best inhibitor in the class of N-acyl derivatives of beta-D-glucopyranosylamine, N-acetyl-beta-D-glucopyranosylamine (1-GlcNAc), has been characterized by kinetic, ultracentrifugation, and crystallographic studies. 1-GlcNAc acts as a competitive inhibitor for both the b (Ki = 32 microM) and the a (Ki = 35 microM) forms of the enzyme with respect to glucose 1-phosphate and in synergism with caffeine, mimicking the binding of glucose. Sedimentation velocity experiments demonstrated that 1-GlcNAc was able to induce dissociation of tetrameric phosphorylase a and stabilization of the dimeric T-state conformation. Co-crystals of the phosphorylase b-1-GlcNAc-IMP complex were grown in space group P4(3)2(1)2, with native-like unit cell dimensions, and the complex structure has been refined to give a crystallographic R factor of 18.1%, for data between 8 and 2.3 A resolution. 1-GlcNAc binds tightly at the catalytic site of T-state phosphorylase b at approximately the same position as that of alpha-D-glucose. The ligand can be accommodated in the catalytic site with very little change in the protein structure and stabilizes the T-state conformation of the 280s loop by making several favorable contacts to Asn 284 of this loop. Structural comparisons show that the T-state phosphorylase b-1-GlcNAc-IMP complex structure is overall similar to the T-state phosphorylase b-alpha-D-glucose complex structure. The structure of the 1-GlcNAc complex provides a rational for the biochemical properties of the inhibitor.
authors.affiliation: Institute of Biological Research and Biotechnology, National Hellenic Research Foundation, Athens, Greece. nikos@apollon.servicenet.ariadne.t.gr
authors.forename: N G
authors.initials: NG
authors.lastname: Oikonomakos
authors.forename: M
authors.initials: M
authors.lastname: Kontou
authors.forename: S E
authors.initials: SE
authors.lastname: Zographos
authors.forename: K A
authors.initials: KA
authors.lastname: Watson
authors.forename: L N
authors.initials: LN
authors.lastname: Johnson
authors.forename: C J
authors.initials: CJ
authors.lastname: Bichard
authors.forename: G W
authors.initials: GW
authors.lastname: Fleet
authors.forename: K R
authors.initials: KR
authors.lastname: Acharya
mesh_terms: D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002110:Caffeine / Q000494:pharmacology; D018360:Crystallography, X-Ray; D004357:Drug Synergism; D004791:Enzyme Inhibitors / Q000737:chemistry*; D005944:Glucosamine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D005947:Glucose / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D015394:Molecular Structure; D006005:Phosphorylases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D011817:Rabbits; D014461:Ultracentrifugation
chemical_list: D004791:Enzyme Inhibitors; D002110:Caffeine; C098108:N-acetylglucopyranosylamine; D006005:Phosphorylases; D005947:Glucose; D005944:Glucosamine