Time-resolved structural studies on catalysis in the crystal with glycogen phosphorylase b

Glycogen phosphorylase catalyses the reversible phosphorylation of glycogen to give glucose-1-phosphate in a reaction mechanism promoted by the 5'-phosphate of the cofactor pyridoxal phosphate. The reaction with the small substrate heptenitol has been probed using Laue diffraction at the Synchrotron Radiation Source, Daresbury. The reaction was initiated following photolysis from a caged phosphate compound 3,5-dinitrophenylphosphate (DNPP). In measurements on photolysis in the crystal using a diode array spectrophotometer approximately 7 mM cage (and hence phosphate) was released from a 21 mM solution with five flashes from a xenon flash lamp. In an experiment with the home source it was shown that DNPP is stable in the crystal under conditions of X-ray measurements and that on flashing sufficient phosphate is released to promote catalysis within 24 h. In a similar experiment with the synchrotron and Laue diffraction, data were recorded before and then 3 min, 15 min and 1 h after initiation of the reaction. Theoretical analysis of the point spread function arising from partial data-sets, numerical calculations with ideal data and the experimental results have shown the importance of low-resolution terms for the interpretation of Laue difference maps. Inclusion of terms obtained from unscrambling the wavelength harmonic overlaps led to significant improvement. The maps showed heptenitol bound at the catalytic site but no evidence for catalysis under these conditions. A rational for the lack of reaction and suggestions for future experiments with improved data are outlined.

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Faculty Opinions recommendation of Three-dimensional structure of phosphorylase kinase at 22 A resolution and its complex with glycogen phosphorylase b.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1004804.55956 ◽

2002 ◽

Author(s):

Esther Bullitt

Keyword(s):

Glycogen Phosphorylase ◽

Three Dimensional ◽

Dimensional Structure ◽

Phosphorylase Kinase ◽

Three Dimensional Structure ◽

Glycogen Phosphorylase B

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A comparison of the activator sites of liver and muscle glycogen phosphorylase b.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)45340-4 ◽

1982 ◽

Vol 257 (23) ◽

pp. 14041-14047

Author(s):

M Kobayashi ◽

G Soman ◽

D J Graves

Keyword(s):

Muscle Glycogen ◽

Glycogen Phosphorylase ◽

Glycogen Phosphorylase B

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Synchrotron radiation macromolecular crystallography: our science and spin offs

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314099896 ◽

2014 ◽

Vol 70 (a1) ◽

pp. C10-C10

Author(s):

John Helliwell

Keyword(s):

Synchrotron Radiation ◽

Protein Complexes ◽

Bound Water ◽

Water Structure ◽

Public Understanding ◽

Anomalous Scattering ◽

Structural Studies ◽

Macromolecular Crystallography ◽

Time Resolved ◽

Chemical Crystallography

I will give an overview of synchrotron radiation (SR) in macromolecular crystallography (MX) instrumentation, methods and applications from the early days to the present, including the evolution of SR sources and on to the `ultimate storage ring'. The build of dedicated beamlines for resonant anomalous scattering, large unit cells, ever smaller crystals and studies up to ultra-high resolution are core benefits. Results include a high output of PDB depositions, the successful use of microcrystals, pushing the frontiers of using high and low photon energies and time-resolved structural studies at even sub-nanosecond resolutions. These intensively physics based developments will be complemented by biological and chemical crystallography research results, encompassing catalysis and marine coloration, as well as the public understanding of our science and its impacts. Spin off benefits include services to the pharmaceutical industry and helping develop chemical crystallography uses of SR. The development of the Laue method with SR has led to pioneering spin off developments in neutron MX, including transfer of the well validated Daresbury Laue software to various neutron facilities worldwide. Neutron MX is gathering pace as new instrumentation and dedicated sample preparation facilities are in place at reactor and spallation neutron sources; smaller samples and much larger molecular weight protein complexes are now feasible for investigation so as to establish their protonation states and bound water structure. With the X-ray lasers, closely linked to the SR developments, we anticipate the use of ever smaller samples such as nanocrystals, nanoclusters and single molecules, as well as opening up femtosecond time-resolved diffraction structural studies. At the SR sources, a very high throughput assessment for the best crystal samples and tackling sub-micron crystals will become widespread.

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Probing the ionization state of substrate α-d-glucopyranosyl phosphate bound to glycogen phosphorylase b

Biochemical Journal ◽

10.1042/bj3081017 ◽

1995 ◽

Vol 308 (3) ◽

pp. 1017-1023 ◽

Cited By ~ 1

Author(s):

I P Street ◽

S G Withers

Keyword(s):

Active Site ◽

Glycogen Phosphorylase ◽

Ph Dependence ◽

Substrate Inhibition ◽

19F Nmr ◽

Ionization State ◽

Nmr Studies ◽

Substrate Analogues ◽

Glycogen Phosphorylase B ◽

Pka Value

The ionization state of the substrate alpha-D-glucopyranosyl phosphate bound at the active site of glycogen phosphorylase has been probed by a number of techniques. Values of Ki determined for a series of substrate analogue inhibitors in which the phosphate moiety bears differing charges suggest that the enzyme will bind both the monoanionic and dianionic substrates with approximately equal affinity. These results are strongly supported by 31P- and 19F-NMR studies of the bound substrate analogues alpha-D-glucopyranosyl 1-methylenephosphonate and 2-deoxy-2-fluoro-alpha-D-glucopyranosyl phosphate, which also suggest that the substrate can be bound in either ionization state. The pH-dependences of the inhibition constants K1 for these two analogues, which have substantially different phosphate pK2 values (7.3 and 5.9 respectively), are found to be essentially identical with the pH-dependence of K(m) values for the substrate, inhibition decreasing according to an apparent pKa value of 7.2. This again indicates that there is no specificity for monoanion or dianion binding and also reveals that binding is associated with the uptake of a proton. As the bound substrate is not protonated, this proton must be taken up by the proton.

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