Binding of beta-D-glucopyranosyl bismethoxyphosphoramidate to glycogen phosphorylase b: kinetic and crystallographic studies |
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Authors: | Chrysina Evangelia D Kosmopoulou Magda N Kardakaris Rozina Bischler Nicolas Leonidas Demetres D Kannan Thanukrishnan Loganathan Duraikkannu Oikonomakos Nikos G |
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Affiliation: | Institute of Organic and Pharmaceutical Chemistry, The National Hellenic Research Foundation, 48 Vassileos Constantinou Ave., 11635 Athens, Greece. |
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Abstract: | In an attempt to identify a new lead molecule that would enable the design of inhibitors with enhanced affinity for glycogen phosphorylase (GP), beta-D-glucopyranosyl bismethoxyphosphoramidate (phosphoramidate), a glucosyl phosphate analogue, was tested for inhibition of the enzyme. Kinetic experiments showed that the compound was a weak competitive inhibitor of rabbit muscle GPb (with respect to alpha-D-glucose-1-phosphate (Glc-1-P)) with a Ki value of 5.9 (+/-0.1) mM. In order to elucidate the structural basis of inhibition, we determined the structure of GPb complexed with the phosphoramidate at 1.83 A resolution. The complex structure reveals that the inhibitor binds at the catalytic site and induces significant conformational changes in the vicinity of this site. In particular, the 280s loop (residues 282-287) shifts 0.4-4.3 A (main-chain atoms) to accommodate the phosphoramidate, but these conformational changes do not lead to increased contacts between the inhibitor and the protein that would improve ligand binding. |
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