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dc.contributor.authorBARFORD, D
dc.contributor.authorSCHWABE, JWR
dc.contributor.authorOIKONOMAKOS, NG
dc.contributor.authorACHARYA, KR
dc.contributor.authorHAJDU, J
dc.contributor.authorPAPAGEORGIOU, AC
dc.contributor.authorMARTIN, JL
dc.contributor.authorKNOTT, JCA
dc.contributor.authorVASELLA, A
dc.contributor.authorJOHNSON, LN
dc.description.abstractRegions 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 /3-glycosidase inhibitor D-gluconohydroximo-1,5-lactone VV-phenylurethane (PUG). Crystallographic binding studies at 2.4-Á 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 Á) to that occupied by other glucosyl compounds bound at the catalytic site. The VV- phenylurethane extends into channel 2 and makes several favorable contacts to the protein that hold the 280-285 loop in place. Kinetic studies show that PUG is a good inhibitor of phosphorylase (K¡ = 0.4 mM) and is directly competitive with the substrate glucose 1-phosphate and noncompetitive with respect to glycogen and AMP. Comparison of the binding interactions between PUG and phosphorylase b with those observed for other glucosyl compounds showed no strong interactions that would allow the catalytic site of phosphorylase to distinguish between the chair or half-chair conformation of the glucopyranose ring. Further comparison of PUG binding with that of uridine diphosphate glucose shows that trigonal geometry at the Cl directs the substituent into channel 2, which is available in the T state, while tetrahedral geometry at Cl directs the substituent into channel 1, which becomes available in the R state. It is concluded that the phosphorylase catalytic site provides no dominant steric factors that promote the distortion of the terminal -D-glucosyl residue from chair to half chair or sofa prior to bond cleavage, a conclusion that is consistent with stereoelectronic theory. It is proposed that the major contribution to the stabilization of the oxocarbonium ion intermediate is mediated through the phosphate of the substrate in a reaction that proceeds by an SN1 mechanism.
dc.publisherAmerican Chemical Society
dc.subject.fieldofresearchMedicinal and biomolecular chemistry
dc.subject.fieldofresearchBiochemistry and cell biology
dc.subject.fieldofresearchBiochemistry and cell biology not elsewhere classified
dc.subject.fieldofresearchMedical biochemistry and metabolomics
dc.titleChannels at the Catalytic Site of Glycogen Phosphorylase b: Binding and Kinetic Studies with the beta-glycosidase Inhibitor D-gluconohydroximo-1,5-lactone N-phenylurethane
dc.typeJournal article
dc.type.descriptionC1 - Articles
dc.type.codeC - Journal Articles
gro.rights.copyrightArchiving not formally supported
gro.hasfulltextNo Full Text
gro.griffith.authorMartin, Jennifer

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