Probing the ionization state of substrate alpha-D-glucopyranosyl phosphate bound to glycogen phosphorylase b.
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.
Street
I P
IP
Department of Chemistry, University of British Columbia, Vancouver, Canada.
Withers
S G
SG
D000818:Animals; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D017611:Fluorine Compounds / Q000378:metabolism / Q000494:pharmacology; D005958:Glucosephosphates / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D006003:Glycogen / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008968:Molecular Conformation; D015394:Molecular Structure; D009132:Muscles / Q000201:enzymology; D006005:Phosphorylases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011485:Protein Binding; D011817:Rabbits
D004791:Enzyme Inhibitors; D017611:Fluorine Compounds; D005958:Glucosephosphates; D006003:Glycogen; C031590:glucose-1-phosphate; D006005:Phosphorylases