Catalytic site of glycogen phosphorylase: Structure of the T state and specificity for α-D-glucose

Stephen R. Sprang, Elizabeth J. Goldsmith, Robert J. Fletterick, Stephen G. Withers, Neil B. Madsen

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Abstract

α-D-Glucose inhibits glycogen phosphorylase a by binding at the catalytic site of the inactive conformer (T state) at the same position as does the substrate α-D-glucose 1-phosphate to the active (R state) enzyme. It is established that recognition of glucose 1-phosphate is highly specific. Here, we show by crystallographic analysis of the α-D-glucose-phosphorylase a complex and by analysis of inhibition by a variety of glucose analogues the nature and specificity of the recognition of the glucosyl group by the T-state enzyme. The catalytic site at which glucose is bound is located at the confluence of the N- and C-terminal domains. Each is an α/β structure consisting of a β-sheet core surrounded by a double tier of α helices. The active-site residues are located on flexible loops of polypeptide chain emanating from the domain boundaries. Glucose participates in at least five well-defined hydrogen bonds with these residues and presents a complementary molecular surface to the active site at the hydrogen-bonded positions of the ligand. Inhibition and model-building studies show that changes in chirality or substitution at any of the glucose hydroxyl groups can abolish or drastically reduce the binding affinity of the ligand. Absence or low activity in glucose analogues can be rationalized as a reduction in hydrogen bonding capacity and/or the introduction of steric conflicts with the enzyme. Although there are substantial differences between the T- and R-state enzymes with respect to active-site conformation, both conformers exhibit specific binding of the glucosyl moiety of α-D-glucose on the one hand (T) and glucose 1-phosphate or half-chair glycosyl analogues (which mimic the proposed carbonium ion intermediates or transition state) on the other (R). A structural interpretation of these observations is presented. By means of inhibition studies with several glucose 1-phosphate analogues and also by inspection of the crystal structure, it is demonstrated that the substrate binding site, in the R-state enzyme, comprises adjacent phosphate and glycosyl subsites. Analogues of the substrate which differ substantially in their carbohydrate moiety demonstrate competitive inhibition by occupation of the phosphate subsite alone.

Original languageEnglish (US)
Pages (from-to)5364-5371
Number of pages8
JournalBiochemistry
Volume21
Issue number21
StatePublished - 1982

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Glycogen Phosphorylase
Catalytic Domain
Glucose
Phosphorylase a
Enzymes
Hydrogen
Hydrogen bonds
Substrates
Phosphates
Ligands
Chirality
Hydrogen Bonding
Occupations
Hydroxyl Radical
Conformations
Substitution reactions
Crystal structure
Inspection
Binding Sites
Carbohydrates

ASJC Scopus subject areas

  • Biochemistry

Cite this

Sprang, S. R., Goldsmith, E. J., Fletterick, R. J., Withers, S. G., & Madsen, N. B. (1982). Catalytic site of glycogen phosphorylase: Structure of the T state and specificity for α-D-glucose. Biochemistry, 21(21), 5364-5371.

Catalytic site of glycogen phosphorylase : Structure of the T state and specificity for α-D-glucose. / Sprang, Stephen R.; Goldsmith, Elizabeth J.; Fletterick, Robert J.; Withers, Stephen G.; Madsen, Neil B.

In: Biochemistry, Vol. 21, No. 21, 1982, p. 5364-5371.

Research output: Contribution to journalArticle

Sprang, SR, Goldsmith, EJ, Fletterick, RJ, Withers, SG & Madsen, NB 1982, 'Catalytic site of glycogen phosphorylase: Structure of the T state and specificity for α-D-glucose', Biochemistry, vol. 21, no. 21, pp. 5364-5371.
Sprang, Stephen R. ; Goldsmith, Elizabeth J. ; Fletterick, Robert J. ; Withers, Stephen G. ; Madsen, Neil B. / Catalytic site of glycogen phosphorylase : Structure of the T state and specificity for α-D-glucose. In: Biochemistry. 1982 ; Vol. 21, No. 21. pp. 5364-5371.
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AB - α-D-Glucose inhibits glycogen phosphorylase a by binding at the catalytic site of the inactive conformer (T state) at the same position as does the substrate α-D-glucose 1-phosphate to the active (R state) enzyme. It is established that recognition of glucose 1-phosphate is highly specific. Here, we show by crystallographic analysis of the α-D-glucose-phosphorylase a complex and by analysis of inhibition by a variety of glucose analogues the nature and specificity of the recognition of the glucosyl group by the T-state enzyme. The catalytic site at which glucose is bound is located at the confluence of the N- and C-terminal domains. Each is an α/β structure consisting of a β-sheet core surrounded by a double tier of α helices. The active-site residues are located on flexible loops of polypeptide chain emanating from the domain boundaries. Glucose participates in at least five well-defined hydrogen bonds with these residues and presents a complementary molecular surface to the active site at the hydrogen-bonded positions of the ligand. Inhibition and model-building studies show that changes in chirality or substitution at any of the glucose hydroxyl groups can abolish or drastically reduce the binding affinity of the ligand. Absence or low activity in glucose analogues can be rationalized as a reduction in hydrogen bonding capacity and/or the introduction of steric conflicts with the enzyme. Although there are substantial differences between the T- and R-state enzymes with respect to active-site conformation, both conformers exhibit specific binding of the glucosyl moiety of α-D-glucose on the one hand (T) and glucose 1-phosphate or half-chair glycosyl analogues (which mimic the proposed carbonium ion intermediates or transition state) on the other (R). A structural interpretation of these observations is presented. By means of inhibition studies with several glucose 1-phosphate analogues and also by inspection of the crystal structure, it is demonstrated that the substrate binding site, in the R-state enzyme, comprises adjacent phosphate and glycosyl subsites. Analogues of the substrate which differ substantially in their carbohydrate moiety demonstrate competitive inhibition by occupation of the phosphate subsite alone.

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