Channels at the catalytic site of glycogen phosphorylase b: binding and kinetic studies with the .beta.-glycosidase inhibitor D-gluconohydroximo-1,5-lactone N-phenylurethane

• Published in: Biochemistry (Easton) Vol. 27; no. 18; pp. 6733 - 6741
• Main Authors: Barford, D, Schwabe, J. W. R, Oikonomakos, N. G, Acharya, K. R, Hajdu, J, Papageorgiou, A. C, Martin, J. L, Knott, J. C. A, Vasella, A, Johnson, L. N
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 06-09-1988
• Subjects: Analytical, structural and metabolic biochemistry; Animals; beta-Glucosidase - antagonists & inhibitors; Binding Sites; Biological and medical sciences; Carbamates - metabolism; Carbamates - pharmacology; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Gluconates - metabolism; Gluconates - pharmacology; Glucosidases - antagonists & inhibitors; In Vitro Techniques; Kinetics; Models, Molecular; Phosphorylase b - antagonists & inhibitors; Phosphorylase b - metabolism; Phosphorylases - metabolism; Protein Conformation; Rabbits; Software; Transferases; Molecular structure; Enzyme; Glycogen phosphorylase; Active site; Glycosidase; Enzyme inhibitor; Glucose; Kinetic parameter; Substrate enzyme complex; X ray diffraction; In vitro; Conformational transition
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00418a014

Regions of low packing density in the vicinity of the catalytic site of glycogen phosphorylase b are described with the aid of a computer program that generates a contour map in which the contour level is inversely proportional to the packing density in the protein. It is shown that, although there is no direct route from the catalytic site to the surface, there are two possible channels that could allow access for substrates following conformational changes in the enzyme. The first channel, channel 1, leads from the catalytic site to the surface close to the nucleoside inhibitor site and requires movements of residues 280-285 and Arg 569 in order to obtain access. Previous crystallographic experiments have shown that in the presence of substrates or R-state inhibitors these parts of the polypeptide chain undergo large conformational changes. The properties of the second channel (channel 2), which is the more extensive channel, have been investigated with the potent beta-glycosidase inhibitor D-gluconohydroximo-1,5-lactone N-phenylurethane (PUG). Crystallographic binding studies at 2.4-A resolution show that the compound binds neatly at the catalytic site of phosphorylase b. The glucopyranosylidene ring, in the half-chair conformation, occupies a similar but not identical position (shift about 0.6 A) to that occupied by other glucosyl compounds bound at the catalytic site.