ROLE OF THE ACTIVE-SITE GATE OF GLYCOGEN-PHOSPHORYLASE IN ALLOSTERIC INHIBITION AND SUBSTRATE-BINDING

The functional role in allosteric regulation of a flexible loop (resid ues 280-288) located near the active site of muscle glycogen phosphory lase was investigated. Mutations were made in residues 283-285 based o n crystallographic studies that indicate that the loop functions as a gate controlling access of substrates to the active site and that thes e specific residues play distinct roles in mimicking the substrate and binding inhibitors when the enzyme is in an inactive conformation, Su bstitution of Ala or Asn for Asp-283, the putative substrate mimic, re sults in a 15-fold decrease in V-max, a 10-fold decrease in the S-0.5 for glucose 1-phosphate, a 10-fold increase in the K-alpha for AMP, an d a 10-20-fold increase in the K-i for glucose, Substitution of Ala fo r Asn-284, which normally forms a hydrogen bond with the inhibitor glu cose, re duces V-max 3-fold, increases the K-i for glucose 2-fold, but has little effect on AMP or glucose 1-phosphate binding or cooperativ ity, Substitution of Asp at 284, on the other hand, reduces V-max 10-f old, elevates the K-i for glucose 10-fold, decreases AMP cooperativity , but has little effect on the affinity of AMP or the cooperativity an d binding of glucose 1-phosphate. Substitution of Leu for Phe-285, whi ch forms aromatic stacking interactions with purine inhibitors, reduce s V-max 2-fold, decreases the affinity for caffeine at least 10-fold, raises the K-alpha for AMP 3-fold, and decreases AMP cooperativity but has little effect on glucose 1-phosphate binding or cooperativity. Th e results of the mutagenesis studies show the importance of the 280's loop for inhibitor binding and modulation of substrate affinity and su ggest a role for the loop in allosteric activation, The propagation of allosteric effects across the domain interface may depend upon specif ic contacts between residues of the 280's loop and the C-terminal doma in.