Thermodynamics of binding of heterobidentate ligands consisting of spacer-connected acarbose and beta-cyclodextrin to the catalytic and starch-binding domains of glucoamylase from Aspergillus niger shows that the catalytic and starch-binding sites are in close proximity in space.
The binding to glucoamylase 1 from Aspergillus niger (GA1) of a series of four synthetic heterobidentate ligands of acarbose and beta-cyclodextrin (beta-CD) linked together has been studied by isothermal titration calorimetry. GA1 consists of a catalytic and a starch-binding domain (SBD) connected by a heavily O-glycosylated linker region. Acarbose is a strong inhibitor of glucoamylase and binds exclusively in the catalytic site, while the cyclic starch mimic beta-CD binds exclusively to the two sites of SBD. No spacer or spacer arms of 14, 36, and 73 A in their extended conformations connect acarbose and beta-CD. These compounds were used as probes for bidentate ligand binding to both domains in order to estimate the distance between the catalytic site and the SBD binding site in solution. DeltaH of binding of the four heterobidentate ligands is within experimental uncertainty equal to the sum of DeltaH of binding of free acarbose and beta-CD, indicating ligand binding to both domains. However, the binding constants are 4-5 orders of magnitude smaller than for the binding of acarbose (K approximately 10(12) M-1), increasing with spacer length from 2 x 10(7) M-1 for no spacer to 1 x 10(8) M-1 for the 73 A spacer. Subsequent titrations with beta-CD of the glucoamylase-bidentate ligand complexes revealed that only one of the two binding sites of SBD was vacant. Further titrations with acarbose to these mixtures showed complete displacement of the acarbose moiety of the bidentate ligands from the catalytic sites. These experiments show that the bidentate ligands bind to both the catalytic domain and SBD. The weakening of the bidentate ligand binding compared to acarbose is a purely entropic effect point to steric hindrance between SBD and the beta-CD moiety. To test this, titrations of glucoamylase 2, a form containing the catalytic domain and the linker region but lacking SBD, with the bidentate ligands were carried out. The results were indistinguishable from the binding of free acarbose. Thus, the reduced affinity of the bidentate ligands observed with GA1 stems from interactions due to SBD. The results show that the catalytic and starch-binding sites are in close proximity in solution and thus indicate considerable flexibility of the linker region.
Department of Biochemistry, August Krogh Institute, University of Copenhagen, Denmark.
B W
BW
Sigurskjold
T
T
Christensen
N
N
Payre
S
S
Cottaz
H
H
Driguez
B
B
Svensson
D020909:Acarbose; D001120:Arginine / Q000737:chemistry; D001234:Aspergillus niger / Q000201:enzymology*; D001665:Binding Sites; D002151:Calorimetry; D002384:Catalysis; D003505:Cyclodextrins / Q000737:chemistry*; D004791:Enzyme Inhibitors / Q000737:chemistry; D005087:Glucan 1,4-alpha-Glucosidase / Q000037:antagonists & inhibitors / Q000737:chemistry*; D006860:Hydrogen Bonding; D008024:Ligands; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D013213:Starch / Q000737:chemistry*; D013816:Thermodynamics; D014312:Trisaccharides / Q000737:chemistry*; D047392:beta-Cyclodextrins
D003505:Cyclodextrins; D004791:Enzyme Inhibitors; D008024:Ligands; D014312:Trisaccharides; D047392:beta-Cyclodextrins; D013213:Starch; D001120:Arginine; D005087:Glucan 1,4-alpha-Glucosidase; C031215:betadex; D020909:Acarbose