Probing the diphosphoglycerate binding pocket of HbA and HbPresbyterian (beta 108Asn --> Lys).
Probing the diphosphoglycerate binding pocket of HbA and HbPresbyterian (beta 108Asn --> Lys).
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DOI：
10.1021/bi9827464
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发表时间：
1999-08
期刊：
影响因子：
2.9
通讯作者：
D. Gottfried;B. Manjula;A. Malavalli;A. Acharya;J. Friedman
中科院分区：
文献类型：
--
作者：
D. Gottfried;B. Manjula;A. Malavalli;A. Acharya;J. Friedman
HbPresbyterian（β 108Asn --> Lys、HbP）在中央空腔中部含有一个额外的正电荷（每个 αβ 二聚体），并且在氯化物存在的情况下表现出比野生型 HbA 更低的氧亲和力。然而，人们对其功能特性改变的分子起源知之甚少。在这项研究中，我们重点关注 Hb 四聚体的 β β 裂口。最近，我们开发了一种使用天然变构效应器 2, 3-二磷酸甘油酸 (DPG) 的荧光类似物来量化 Hb 中央腔 β 端配体结合亲和力的方法 [Gottfried, D. S., et al. (1997) J.Biol。化学。 272, 1571-1578]。时间相关的单光子计数荧光寿命研究用于评估酸性和中性 pH 条件下脱氧和 CO 连接状态下芘四磺酸盐与 HbA 和 HbP 的结合。天然蛋白和突变蛋白均在弱结合位点和强结合位点结合探针；在所有情况下，与 HbP 的结合都强于与 HbA 的结合。最引人注目的发现是，对于 HbA，结合亲和力变化如下：脱氧 (pH 6.35) > 脱氧 (pH 7.20) > CO (pH 6.35)；然而，与 HbP 的结合与连接或 pH 无关。突变氧蛋白还通过与β-β裂的His残基相连的可逆酰基咪唑途径水解对硝基苯乙酸酯，其水解速率比HbA高得多。这意味着 DPG 结合口袋处这些残基的微环境受到扰动。由于中央空腔中间存在新的正电荷而产生的结构后果已传递到蛋白质的 β β 裂口，即使在其配体构象中也是如此。这与新描述的配体 HbPresbyterian 的四元态 (B) 以及变构控制机制的相关变化一致。
HbPresbyterian (beta 108Asn --> Lys, HbP) contains an additional positive charge (per alpha beta dimer) in the middle of the central cavity and exhibits a lower oxygen affinity than wild-type HbA in the presence of chloride. However, very little is known about the molecular origins of its altered functional properties. In this study, we have focused on the beta beta cleft of the Hb tetramer. Recently, we developed an approach for quantifying the ligand binding affinity to the beta-end of the Hb central cavity using fluorescent analogues of the natural allosteric effector 2, 3-diphosphoglycerate (DPG) [Gottfried, D. S., et al. (1997) J. Biol. Chem. 272, 1571-1578]. Time-correlated single-photon counting fluorescence lifetime studies were used to assess the binding of pyrenetetrasulfonate to both HbA and HbP in the deoxy and CO ligation states under acidic and neutral pH conditions. Both the native and mutant proteins bind the probe at a weak binding site and a strong binding site; in all cases, the binding to HbP was stronger than to HbA. The most striking finding was that for HbA the binding affinity varies as follows: deoxy (pH 6.35) > deoxy (pH 7.20) > CO (pH 6.35); however, the binding to HbP is independent of ligation or pH. The mutant oxy protein also hydrolyzes p-nitrophenyl acetate, through a reversible acyl-imidazole pathway linked to the His residues of the beta beta cleft, at a considerably higher rate than does HbA. This implies a perturbation of the microenvironment of these residues at the DPG binding pocket. Structural consequences due to the presence of the new positive charge in the middle of the central cavity have been transmitted to the beta beta cleft of the protein, even in its liganded conformation. This is consistent with a newly described quaternary state (B) for liganded HbPresbyterian and an associated change in the allosteric control mechanism.