Arnone A, Rogers P H, Benesch R E, Benesch R, Kwong S
J Biol Chem. 1986 May 5;261(13):5853-7.
Previous studies have shown that pteroylheptaglutamate (PteGlu7) can form a 1:1 complex with deoxyhemoglobin. The solution and crystallographic studies reported in this paper delineate the nature of the PteGlu7 binding site. We find that the three structural elements of PteGlu7 (the pteridine moiety, the p-aminobenzoyl portion, and the glutamate groups) each contribute to the binding energy by interacting with residues in the central cavity between the beta subunits and with residues at the alpha 1 beta 1 interface. Identification of the 2,3-diphosphoglycerate (DPG) binding site as part of the PteGlu7 binding site was accomplished in two ways; first by the demonstration of reduced PteGlu7 binding to hemoglobin selectively modified by pyridoxylation at this site, and second by the finding that DPG and PteGlu7 bind to deoxyhemoglobin in a competitive manner. In addition, since analogs of PteGlu7 in which the pteridine moiety is modified display reduced binding, it can be concluded that the pteridine group also contributes significantly to the binding energy. The crystallographic studies are completely consistent with the results determined in solution. A difference electron density image at 4.3 A resolution shows that the pteridine and p-aminobenzoyl groups are nestled against an interior edge of the alpha 1 beta 1 interface with the pteridine ring interacting with Phe 36 alpha 1 and the p-aminobenzoyl group positioned against a portion of the H helix between residues Lys 132 beta 1 and Ala 135 beta 1. The difference density for the glutamate residues is less well resolved (for reasons described in the text), but it is clear that some of the carboxylate side chains must interact with residues at the DPG binding site.
先前的研究表明，蝶酰庚谷氨酸（PteGlu7）可与脱氧血红蛋白形成1:1复合物。本文报道的溶液和晶体学研究描述了PteGlu7结合位点的性质。我们发现，PteGlu7的三个结构元件（蝶啶部分、对氨基苯甲酰部分和谷氨酸基团）各自通过与β亚基之间中心腔内的残基以及α1β1界面处的残基相互作用，对结合能有所贡献。通过两种方式确定了2,3 - 二磷酸甘油酸（DPG）结合位点是PteGlu7结合位点的一部分；首先，通过证明PteGlu7与在此位点经吡哆醛化选择性修饰的血红蛋白的结合减少，其次，通过发现DPG和PteGlu7以竞争方式结合到脱氧血红蛋白上。此外，由于蝶啶部分被修饰的PteGlu7类似物显示出结合减少，可以得出结论，蝶啶基团对结合能也有显著贡献。晶体学研究与溶液中测定的结果完全一致。分辨率为4.3 Å的差分电子密度图像显示，蝶啶和对氨基苯甲酰基团紧靠α1β1界面的内部边缘，蝶啶环与Phe 36α1相互作用，对氨基苯甲酰基团位于β1亚基残基Lys 132和Ala 135之间的H螺旋的一部分上。谷氨酸残基的差分密度分辨率较差（原因在文中描述），但很明显一些羧基侧链必须与DPG结合位点处的残基相互作用。
