The binding of D-gluconohydroximo-1,5-lactone to glycogen phosphorylase. Kinetic, ultracentrifugation and crystallographic studies.
Combined kinetic, ultracentrifugation and X-ray-crystallographic studies have characterized the effect of the beta-glucosidase inhibitor gluconohydroximo-1,5-lactone on the catalytic and structural properties of glycogen phosphorylase. In the direction of glycogen synthesis, gluconohydroximo-1,5-lactone was found to competitively inhibit both the b (Ki 0.92 mM) and the alpha form of the enzyme (Ki 0.76 mM) with respect to glucose 1-phosphate in synergism with caffeine. In the direction of glycogen breakdown, gluconohydroximo-1,5-lactone was found to inhibit phosphorylase b in a non-competitive mode with respect to phosphate, and no synergism with caffeine could be demonstrated. Ultracentrifugation and crystallization experiments demonstrated that gluconohydroximo-1,5-lactone was able to induce dissociation of tetrameric phosphorylase alpha and stabilization of the dimeric T-state conformation. A crystallographic binding study with 100 mM-gluconohydroximo-1,5-lactone at 0.24 nm (2.4 A) resolution showed a major peak at the catalytic site, and no significant conformational changes were observed. Analysis of the electron-density map indicated that the ligand adopts a chair conformation. The results are discussed with reference to the ability of the catalytic site of the enzyme to distinguish between two or more conformations of the glucopyranose ring.
Papageorgiou
A C
AC
Biological Research Center, National Hellenic Research Foundation, Athens, Greece.
Oikonomakos
N G
NG
Leonidas
D D
DD
Bernet
B
B
Beer
D
D
Vasella
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002219:Carbamates / Q000737:chemistry; D005942:Gluconates / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D006860:Hydrogen Bonding; D007097:Imines / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology; D010763:Phosphorylase b / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D011817:Rabbits; D014461:Ultracentrifugation / Q000379:methods; D014961:X-Ray Diffraction / Q000379:methods
D002219:Carbamates; D005942:Gluconates; D007097:Imines; C057814:gluconohydroximo-1,5-lactone-N-phenylurethane; C067867:gluconohydroximo-1,5-lactone; D010763:Phosphorylase b