N‐acetyl‐β‐D‐glucopyranosylamine: A potent T‐state inhibitor of glycogen phosphorylase. A comparison with α‐D‐glucose

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 α‐D‐glucose (Ki = 1.7 mM). The best inhibitor in the class of N‐acyl derivatives of β‐D‐glucopyranosylamine, N‐acetyl‐β‐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 μM) and the α (Ki = 35 μM) 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 α and stabilization of the dimeric T‐state conformation. Co‐crystals of the phosphorylase b‐1‐GlcNAc‐IMP complex were grown in space group P43212, 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 Å resolution. 1‐GlcNAc binds tightly at the catalytic site of T‐state phosphorylase b at approximately the same position as that of α‐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‐α‐D‐glucose complex structure. The structure of the 1‐GlcNAc complex provides a rational for the biochemical properties of the inhibitor.

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