Kinetic properties of tetrameric glycogen phosphorylase <i>b</i> in solution and in the crystalline state

Leonidas, D.D.; Oikonomakos, Nikos G.; Papageorgiou, Anastassios C.; Sotiroudis, Theodore G.

doi:10.1002/pro.5560010906

Cited by 10 publications

(9 citation statements)

References 61 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…3C, upper panel) that was not significantly different from heats of dilution and mixing determined in controls, indicating that 10 μM CP‐316819 was sufficient to completely block the interaction of the 16mer G L peptide with 25 μM phosphorylase a . Although in vivo muscle phosphorylase a is a dimer, in vitro at 25 μM phosphorylase a may be mainly tetrameric [11,12]. The ratio, CP‐316819: phosphorylase a monomer of 0.4:1 that blocks interaction with G L suggests that homotropic cooperative effects between the subunits of phosphorylase a allow inhibition of approximately two subunits in the tetramer (or one subunit in the dimer) to allosterically change the conformation of the other subunits(s) to the inhibited structure [13].…”

Section: Resultsmentioning

confidence: 99%

The hepatic PP1 glycogen‐targeting subunit interaction with phosphorylase a can be blocked by C‐terminal tyrosine deletion or an indole drug

et al. 2007

View full text Add to dashboard Cite

The inhibition of hepatic glycogen-associated protein phosphatase-1 (PP1-G L ) by glycogen phosphorylase a prevents the dephosphorylation and activation of glycogen synthase, suppressing glycogen synthesis when glycogenolysis is activated. Here, we show that a peptide ( 280 LGPYY 284 ) comprising the last five amino acids of G L retains high-affinity interaction with phosphorylase a and that the two tyrosines play crucial roles. Tyr284 deletion abolishes binding of phosphorylase a to G L and replacement by phenylalanine is insufficient to restore high-affinity binding. We show that a phosphorylase inhibitor blocks the interaction of phosphorylase a with the G L C-terminus, suggesting that the latter interaction could be targeted to develop an antidiabetic drug.

show abstract

Section: Resultsmentioning

confidence: 99%

The hepatic PP1 glycogen‐targeting subunit interaction with phosphorylase a can be blocked by C‐terminal tyrosine deletion or an indole drug

et al. 2007

View full text Add to dashboard Cite

show abstract

“…These changes include movements in part of the glycogen storage site. The R state crystals have been shown to be active with kinetic similar properties to those of the enzyme in solution (Leonidas et al, 1992). R state crystals of both GPb and GPa grown in the presence of ammonium sulfate have a sulfate ion bound at the catalytic site.…”

Section: Kineticsmentioning

confidence: 94%

Ternary Complex Crystal Structures of Glycogen Phosphorylase with the Transition State Analogue Nojirimycin Tetrazole and Phosphate in the T and R States^,

et al. 1996

Self Cite

View full text Add to dashboard Cite

Catalysis by glycogen phosphorylase involves a mechanism in which binding of one substrate tightens the binding of the other substrate to produce a productive ternary enzyme-substrate complex. In this work the molecular basis for this synergism is probed in crystallographic studies on ternary complexes in which the glucosyl component is substituted by the putative transition state analogue nojirimycin tetrazole, a compound which has been established previously as a transition state analogue inhibitor for a number of glycosidases. Kinetic studies with glycogen phosphorylase showed that nojirimycin tetrazole is a competitive inhibitor with respect to glucose 1-phosphate and uncompetitive with respect to phosphate. Ki values for the phosphorylase-AMP-glycogen complex and the phosphorylase-AMP-glycogen-phosphate complexes are 700 microM and 53 microM, respectively, indicating that by itself norjirimycin tetrazole has poor affinity for glycogen phosphorylase but that phosphate substantially improves the binding of norjirimycin tetrazole. X-ray crystallographic binding studies to 2.4 A resolution with T state phosphorylase b crystals showed that nojirimycin tetrazole binds at the catalytic site and promotes the binding of phosphate through direct interactions. Phosphate binding is accompanied by conformational changes that bring a crucial arginine (Arg569) into the catalytic site. The positions of the phosphate oxygens were definitively established in X-ray crystallographic binding experiments at 100 K to 1.7 A resolution using synchrotron radiation. X-ray crystallographic binding studies at 2.5 A resolution with R state glycogen phosphorylase crystals showed that the protein atoms and water molecules in contact with the nojirimycin tetrazole and the phosphate are similar to those in the T state. In both T and R states the phosphate ion is within hydrogen-bonding distance of the cofactor pyridoxal 5'-phosphate group and in ionic contact with the N-1 atom of the tetrazole ring. The results are consistent with previous time-resolved structural studies on complexes with heptenitol and phosphate. The structural and kinetic results suggest that nojirimycin tetrazole in combination with phosphate exhibits properties consistent with a transition state analogue and demonstrate how one promotes the binding of the other.

show abstract

“…The enzyme (10-I00 ,ug/ml) was preincubated with glycogen for 15 min at 30°C before the reaction was started by adding the glucose 1-phosphate. Under these assay conditions, the enzyme exists as a dimer [2]. Samples were withdrawn at 1 min intervals over the period from 1-5 min and transferred into 0.2 % SDS to stop the reaction.…”

Section: Determination Of Phosphorylase Activitymentioning

confidence: 99%

“…Its phosphorylated and unphosphorylated forms are known as phosphorylases a and b respectively. The enzyme is active as a dimer (Mr = 195000) but may also form tetramers that still retain some activity [1,2]. The structure of the R-state tetrameric phosphorylase b, based upon the X-ray analysis of crystals of the enzyme grown from ammonium sulphate solution, has been solved at 2.8 A (1 A = 10-10 m) [3,4].…”

Section: Introductionmentioning

confidence: 99%

Sulphate-activated phosphorylase b: the pH-dependence of catalytic activity

Zographos

Oikonomakos

Dixon

et al. 1995

Biochemical Journal

View full text Add to dashboard Cite

The pH-dependence of sulphate-activated phosphorylase b has been studied in the direction of glycogen synthesis. The bell-shaped curve of the pH-dependence of the catalytic constant for the AMP-activated enzyme showed pK values of 6.1 and 7.3, but the curve for the enzyme activated by 0.9 M ammonium sulphate showed a drop of activity on the acid side at much higher pH values. Its bell was centred at pH 7.8 but it was too narrow to be characterized by only two pK values. The narrowness of the curve could be explained by positive co-operativity, but not its unusually steep acid side. We suggest that the fall on the acid side is due to more than one hydronation (addition of H+). The points can be fitted by a curve with two de-activating hydronations and a de-activating dehydronation having identical titration pK values of 7.5, and hence molecular values of 7.0, 7.5 and 8.0. If both 0.9 M ammonium sulphate and 5 mM AMP are added, the bell is as broad as with AMP alone, but is somewhat raised in pH optimum. The results are discussed in the light of new structural data from crystallographic studies on binary complexes of the enzyme.

show abstract

Kinetic properties of tetrameric glycogen phosphorylase b in solution and in the crystalline state

Cited by 10 publications

References 61 publications

The hepatic PP1 glycogen‐targeting subunit interaction with phosphorylase a can be blocked by C‐terminal tyrosine deletion or an indole drug

The hepatic PP1 glycogen‐targeting subunit interaction with phosphorylase a can be blocked by C‐terminal tyrosine deletion or an indole drug

Ternary Complex Crystal Structures of Glycogen Phosphorylase with the Transition State Analogue Nojirimycin Tetrazole and Phosphate in the T and R States^,

Sulphate-activated phosphorylase b: the pH-dependence of catalytic activity

Contact Info

Product

Resources

About

Kinetic properties of tetrameric glycogen phosphorylase b in solution and in the crystalline state

Cited by 10 publications

References 61 publications

The hepatic PP1 glycogen‐targeting subunit interaction with phosphorylase a can be blocked by C‐terminal tyrosine deletion or an indole drug

The hepatic PP1 glycogen‐targeting subunit interaction with phosphorylase a can be blocked by C‐terminal tyrosine deletion or an indole drug

Ternary Complex Crystal Structures of Glycogen Phosphorylase with the Transition State Analogue Nojirimycin Tetrazole and Phosphate in the T and R States,

Sulphate-activated phosphorylase b: the pH-dependence of catalytic activity

Contact Info

Product

Resources

About

Ternary Complex Crystal Structures of Glycogen Phosphorylase with the Transition State Analogue Nojirimycin Tetrazole and Phosphate in the T and R States^,