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.
Isolation and characterization of glycogen phosphorylase b from chicken breast muscle: comparison with a protein extracted from the M. line.