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-cyclod extrin (beta-CD) linked together has been studied by isothermal titrat ion calorimetry. GA1 consists of a catalytic and a starch-binding doma in (SBD) connected by a heavily O-glycosylated linker region. Acarbose is a strong inhibitor of glucoamylase and binds exclusively in the ca talytic 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 An gstrom in their extended conformations connect acarbose and beta-CD Th ese 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. Delta H of binding of the four h eterobidentate ligands is within experimental uncertainty equal to the sum of Delta H of binding of free acarbose and beta-CD, indicating li gand binding to both domains. However, the binding constants are 4-5 o rders of magnitude smaller than for the binding of acarbose (K approxi mate to 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 Angstrom 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. Fu rther titrations with acarbose to these mixtures showed complete displ acement of the acarbose moiety of the bidentate ligands from the catal ytic sites. These experiments show that the bidentate ligands bind to both the catalytic domain and SBD. The weakening of the bidentate liga nd binding compared to acarbose is a purely entropic effect point to s teric hindrance between SBD and the beta-CD moiety. To test this, titr ations of glucoamylase 2, a form containing the catalytic domain and t he linker region but lacking SBD, with the bidentate ligands were carr ied out. The results were indistinguishable from the binding of free a carbose. 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.