Sigurskjold B W, Christensen T, Payre N, Cottaz S, Driguez H, Svensson B
Department of Biochemistry, August Krogh Institute, University of Copenhagen, Denmark.
Biochemistry. 1998 Jul 21;37(29):10446-52. doi: 10.1021/bi9807310.
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.
通过等温滴定量热法研究了一系列由阿卡波糖和β-环糊精（β-CD）连接在一起的四种合成异双齿配体与黑曲霉葡糖淀粉酶1（GA1）的结合情况。GA1由一个催化结构域和一个淀粉结合结构域（SBD）组成，二者通过一个高度O-糖基化的连接区相连。阿卡波糖是葡糖淀粉酶的一种强抑制剂，仅结合在催化位点，而环状淀粉模拟物β-CD仅结合在SBD的两个位点。在其伸展构象中，不存在14、36和73 Å的间隔基或间隔臂连接阿卡波糖和β-CD。这些化合物被用作双齿配体与两个结构域结合的探针，以估计溶液中催化位点和SBD结合位点之间的距离。四种异双齿配体的结合焓在实验不确定度范围内等于游离阿卡波糖和β-CD结合焓的总和，表明配体与两个结构域都发生了结合。然而，结合常数比阿卡波糖的结合常数（K约为10¹² M⁻¹）小4 - 5个数量级，随着间隔基长度从无间隔基时的2×10⁷ M⁻¹增加到73 Å间隔基时的1×10⁸ M⁻¹。随后用β-CD滴定葡糖淀粉酶 - 双齿配体复合物，结果显示SBD的两个结合位点中只有一个是空的。再用阿卡波糖滴定这些混合物，结果表明双齿配体的阿卡波糖部分从催化位点完全被取代。这些实验表明双齿配体与催化结构域和SBD都发生了结合。与阿卡波糖相比，双齿配体结合的减弱是一种纯粹的熵效应，表明SBD和β-CD部分之间存在空间位阻。为了验证这一点，用双齿配体滴定了葡糖淀粉酶2（一种包含催化结构域和连接区但缺乏SBD的形式）。结果与游离阿卡波糖的结合情况没有区别。因此，用GA1观察到的双齿配体亲和力降低源于SBD引起的相互作用。结果表明，在溶液中催化位点和淀粉结合位点靠得很近，因此表明连接区具有相当大的灵活性。
