The functional role in allosteric regulation of a flexible loop (residues 280-288) located near the active site of muscle glycogen phosphorylase was investigated. Mutations were made in residues 283-285 based on crystallographic studies that indicate that the loop functions as a gate controlling access of substrates to the active site and that these specific residues play distinct roles in mimicking the substrate and binding inhibitors when the enzyme is in an inactive conformation. Substitution of Ala or Asn for Asp-283, the putative substrate mimic, results in a 15-fold decrease in Vmax, a 10-fold decrease in the S0.5 for glucose 1-phosphate, a 10-fold increase in the Ka for AMP, and a 10-20-fold increase in the Ki for glucose. Substitution of Ala for Asn-284, which normally forms a hydrogen bond with the inhibitor glucose, reduces Vmax 3-fold, increases the Ki for glucose 2-fold, but has little effect on AMP or glucose 1-phosphate binding or cooperativity. Substitution of Asp at 284, on the other hand, reduces Vmax 10-fold, elevates the Ki for glucose 10-fold, decreases AMP cooperativity, but has little effect on the affinity of AMP or the cooperativity and binding of glucose 1-phosphate. Substitution of Leu for Phe-285, which forms aromatic stacking interactions with purine inhibitors, reduces Vmax 2-fold, decreases the affinity for caffeine at least 10-fold, raises the Ka for AMP 3-fold, and decreases AMP cooperativity but has little effect on glucose 1-phosphate binding or cooperativity. The results of the mutagenesis studies show the importance of the 280's loop for inhibitor binding and modulation of substrate affinity and suggest a role for the loop in allosteric activation. The propagation of allosteric effects across the domain interface may depend upon specific contacts between residues of the 280's loop and the C-terminal domain.