N-ACETYL-BETA-D-GLUCOPYRANOSYLAMINE - A POTENT T-STATE INHIBITOR OF GLYCOGEN-PHOSPHORYLASE - A COMPARISON WITH ALPHA-D-GLUCOSE

Structure-based drug design has led to the discovery of a number of gl ucose analogue inhibitors of glycogen phosphorylase that have an incre ased affinity compared to alpha-D-glucose (K-i = 1.7 mM). The best inh ibitor in the class of N-acyl derivatives of beta-D-glucopyranosylamin e, N-acetyl-beta-D-glucopyranosylamine (1-GlcNAc), has been characteri zed by kinetic, ultracentrifugation, and crystallographic studies. 1-G lcNAc acts as a competitive inhibitor for both the b (K-i = 32 mu M) a nd the a (K-i = 35 mu M) forms of the enzyme with respect to glucose 1 -phosphate and in synergism with caffeine, mimicking the binding of gl ucose. Sedimentation velocity experiments demonstrated that 1-GlcNAc w as able to induce dissociation of tetrameric phosphorylase a and stabi lization of the dimeric T-state conformation. Go-crystals of the phosp horylase 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 b een refined to give a crystallographic R factor of 18.1%, for data bet ween 8 and 2.3 Angstrom resolution. 1-GlcNAc binds tightly at the cata lytic site of T-state phosphorylase b at approximately the same positi on as that of alpha-D-glucose. The ligand can be accommodated in the c atalytic site with very little change in the protein structure and sta bilizes the T-state conformation of the 280s loop by making several fa vorable contacts to Asn 284 of this loop. Structural comparisons show that the T-state phosphorylase b-1-GlcNAc-IMP complex structure is ove rall similar to the T-state phosphorylase b-alpha-D-glucose complex st ructure. The structure of the 1-GlcNAc complex provides a rational for the biochemical properties of the inhibitor.