Uridine(5')diphospho(1)-alpha-D-glucose. A binding study to glycogen phosphorylase b in the crystal.
UDP-glucose is an R-state inhibitor of glycogen phosphorylase b, competitive with the substrate, glucose 1-phosphate and noncompetitive with the allosteric activator, AMP. Diffusion of 100 mM UDP-glucose into crystals of phosphorylase b resulted in a difference Fourier synthesis at 0.3-nm resolution that showed two peaks: (a) binding at the allosteric site and (b) binding at the catalytic site. At the allosteric site the whole of the UDP-glucose molecule can be located. It is in a well defined folded conformation with its uracil portion in a similar position to that observed for the adenine of AMP. The uracil and the glucose moieties stack against the aromatic side chains of Tyr-75 and Phe-196, respectively. The phosphates of the pyrophosphate component interact with Arg-242, Arg-309 and Arg-310. At the catalytic site, the glucose-1-P component of UDP-glucose is firmly bound in a position similar to that observed for glucose 1-phosphate. The pyrophosphate is also well located with the glucose phosphate interacting with the main-chain NH groups at the start of the glycine-loop alpha helix and the uridine phosphate interacting through a water molecule with the 5'-phosphate of the cofactor pyridoxal phosphate and with the side chains of residues Tyr-573, Lys-574 and probably Arg-569. However the position of the uridine cannot be located although analysis by thin-layer chromatography showed that no degradation had taken place. Binding of UDP-glucose to the catalytic site promotes extensive conformational changes. The loop 279-288 which links the catalytic site to the nucleoside inhibitor site is displaced and becomes mobile. Concomitant movements of residues His-571, Arg-569, and the loop 378-383, together with the major loop displacement, result in an open channel to the catalytic site. Comparison with other structural results shows that these changes form an essential feature of the T to R transition. They allow formation of the phosphate recognition site at the catalytic site and destroy the nucleoside inhibitor site. Kinetic experiments demonstrate that UDP-glucose activates the enzyme in the presence of high concentrations of the weak activator IMP, because of its ability to decrease the affinity of IMP for the inhibitor site.
Oikonomakos
N G
NG
Laboratory and Molecular Biophysics, Oxford, England.
Acharya
K R
KR
Stuart
D I
DI
Melpidou
A E
AE
McLaughlin
P J
PJ
Johnson
L N
LN
D000495:Allosteric Site; D001667:Binding, Competitive; D002384:Catalysis; D055598:Chemical Phenomena; D002621:Chemistry; D004789:Enzyme Activation / Q000187:drug effects; D006860:Hydrogen Bonding; D007291:Inosine Monophosphate / Q000494:pharmacology; D008958:Models, Molecular; D006005:Phosphorylases / Q000037:antagonists & inhibitors*; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D014532:Uridine Diphosphate Glucose / Q000378:metabolism / Q000494:pharmacology*; D014539:Uridine Diphosphate Sugars / Q000494:pharmacology*; D014961:X-Ray Diffraction
D014539:Uridine Diphosphate Sugars; D007291:Inosine Monophosphate; D006005:Phosphorylases; D014532:Uridine Diphosphate Glucose