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Volume 271, Number 37, Issue of September 13, 1996 pp. 22305-22309
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Role of the Active Site Gate of Glycogen Phosphorylase in Allosteric Inhibition and Substrate Binding

(Received for publication, April 26, 1996, and in revised form, June 11, 1996)

From the Department of Biochemistry and Biophysics, University of California, San Francisco, California 94143-0448

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 V_max, a 10-fold decrease in the S_0.5 for glucose 1-phosphate, a 10-fold increase in the K_a for AMP, and a 10-20-fold increase in the K_i for glucose. Substitution of Ala for Asn-284, which normally forms a hydrogen bond with the inhibitor glucose, reduces V_max 3-fold, increases the K_i 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 V_max 10-fold, elevates the K_i 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 V_max 2-fold, decreases the affinity for caffeine at least 10-fold, raises the K_a 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.

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