Subunit interactions and their relation to the allosteric properties of rabbit skeletal muscle phosphorylase b

Kastenschmidt, L. L.; Kastenschmidt, Jeannine; Helmreich, Ernst

doi:10.1021/bi00850a037

Cited by 272 publications

(126 citation statements)

References 50 publications

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“…GPa was prepared and recrystallized as described (23). Protein concentration was determined from absorbance measurements at 280 nm using an absorbance index A 1 cm 1% ϭ 13.2 (26). Glucose-1-P (dipotassium salt), AMP, (oyster) glycogen, and other chemicals were obtained from Sigma.…”

Section: Kinetic Experimentsmentioning

confidence: 99%

Flavopiridol Inhibits Glycogen Phosphorylase by Binding at the Inhibitor Site

Oikonomakos¹,

Schnier²,

Zographos³

et al. 2000

Journal of Biological Chemistry

122

111

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Section: Kinetic Experimentsmentioning

confidence: 99%

Flavopiridol Inhibits Glycogen Phosphorylase by Binding at the Inhibitor Site

Oikonomakos¹,

Schnier²,

Zographos³

et al. 2000

Journal of Biological Chemistry

122

111

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“…Bound nucleotides were removed from the enzyme as previously described (Melpidou & Oikonomakos, 1983). Protein concentration was determined from absorbance measurements at 280 nm using an absorbance index A;:,,, = 13.2 (Kastenschmidt et al, 1968).…”

Section: Preparation Of Phosphorylasementioning

confidence: 99%

N‐acetyl‐β‐D‐glucopyranosylamine: A potent T‐state inhibitor of glycogen phosphorylase. A comparison with α‐D‐glucose

et al. 1995

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Structure-based drug design has led to the discovery of a number of glucose analogue inhibitors of glycogen phosphorylase that have an increased affinity compared to a-D-glucose (Ki = 1.7 mM). The best inhibitor in the class of N-acyl derivatives of P-D-glucopyranosylamine, N-acetyl-P-D-glucopyranosylamine (I-GlcNAc), has been characterized by kinetic, ultracentrifugation, and crystallographic studies. 1-GlcNAc acts as a competitive inhibitor for both the b (K, = 32 pM) and the a ( K , = 35 PM) forms of the enzyme with respect to glucose I-phosphate and in synergism with caffeine, mimicking the binding of glucose. Sedimentation velocity experiments demonstrated that 1-GlcNAc was able to induce dissociation of tetrameric phosphorylase a and stabilization of the dimeric T-state conformation. Co-crystals of the phosphorylase b-I-GlcNAc-IMP complex were grown in space group P432,2, with native-like unit cell dimensions, and the complex structure has been refined to give a crystallographic R factor of 18.1 %, for data between 8 and 2.3 A resolution. I-GlcNAc binds tightly at the catalytic site of T-state phosphorylase b at approximately the same position as that of a-D-glucose. The ligand can be accommodated in the catalytic site with very little change in the protein structure and stabilizes the T-state conformation of the 280s loop by making several favorable contacts to Asn 284 of this loop. Structural comparisons show that the T-state phosphorylase b-I-GlcNAc-IMP complex structure is overall similar to the T-state phosphorylase b-a-D-glucose complex structure. The structure of the I-GlcNAc complex provides a rational for the biochemical properties of the inhibitor.

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“…Rabbit muscle GPb was purified according to Fischer and Krebs [34]. The concentrations of GPb and PhK were determined from absorbance measurements at 280 nm using extinction coefficients ε 1% 1cm =13.2 [35] and ε 1% 1cm =12.4 [36], respectively. PhK-γtrnc concentration was determined according to Bradford [37].…”

Section: Methodsmentioning

confidence: 99%

An evaluation of indirubin analogues as phosphorylase kinase inhibitors

Begum

Skamnaki

Moffatt

et al. 2015

Journal of Molecular Graphics and Modelling

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Phosphorylase kinase (PhK) has been linked with a number of conditions such as glycogen storage diseases, psoriasis, type 2 diabetes and more recently, cancer (Camus S. et al., Oncogene 2012, 31, 4333). However, with few reported structural studies on PhK inhibitors, this hinders a structure based drug design approach. In this study, the inhibitory potential of 38 indirubin analogues have been investigated. 11 of these ligands had IC50 values in the range 0.170 -0.360 µM, with indirubin-3'-acetoxime (1c) the most potent. 7-bromoindirubin-3'-oxime (13b), an antitumor compound which induces caspase-independent cell-death (Ribas J. et al., Oncogene, 2006, 25, 6304) is revealed as a specific inhibitor of PhK (IC50 = 1.8 µM). Binding assay experiments performed using both PhK-holo and PhK-γtrnc confirmed the inhibitory effects to arise from binding at the kinase domain (γ subunit). High level computations using QM/MM-PBSA binding free energy calculations were in good agreement with experimental binding data, as determined using statistical analysis, and support binding at the ATP-binding site. The value of a QM description for the binding of halogenated ligands exhibiting σ-hole effects is highlighted. A new statistical metric, the 'sum of the modified logarithm of ranks' (SMLR), has been defined which measures performance of a model for both the "early recognition" (ranking earlier/higher) of active compounds and their relative ordering by potency. Through a detailed structure activity relationship analysis considering other kinases (CDK2, CDK5 and GSK-3α/β), 6'(Z) and 7(L) indirubin substitutions have been identified to achieve selective PhK inhibition. The key PhK binding site residues involved can also be targeted using other ligand scaffolds in future work.

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Subunit interactions and their relation to the allosteric properties of rabbit skeletal muscle phosphorylase b

Abstract: Work was initiated to clarify the role of subunit interactions in the allosteric transitions of rabbit skeletal muscle phosphorylase b. When four SH groups per mole of phosphorylase dimer b are blocked by treating the enzyme with 5 3 '-dithiobis(2-

Cited by 272 publications

References 50 publications

Flavopiridol Inhibits Glycogen Phosphorylase by Binding at the Inhibitor Site

Flavopiridol Inhibits Glycogen Phosphorylase by Binding at the Inhibitor Site

N‐acetyl‐β‐D‐glucopyranosylamine: A potent T‐state inhibitor of glycogen phosphorylase. A comparison with α‐D‐glucose

An evaluation of indirubin analogues as phosphorylase kinase inhibitors

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