Protein-ligand interactions as a driving force for a high-enthalpy two-state transition in glutamate dehydrogenase: the opposing roles of phosphate and acetate ions.
Protein-ligand interactions as a driving force for a high-enthalpy two-state transition in glutamate dehydrogenase: the opposing roles of phosphate and acetate ions.
复制标题
蛋白质-配体相互作用作为谷氨酸脱氢酶高焓二态转变的驱动力：磷酸盐和乙酸盐离子的相反作用。
DOI：
10.1006/abbi.1994.1277
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发表时间：
1994
影响因子：
3.9
通讯作者：
Fisher,HF
中科院分区：
文献类型：
--
作者：
Singh,N;Fisher,HF
已知还原辅酶(NADPH)与牛肝谷氨酸脱氢酶的结合受磷酸盐、醋酸盐和其他阴离子的存在以及介质的pH的控制。这些效应器通过降低酶上可电离基团的pK(8.5)来调节这种结合，这种pK漂移与蛋白质中的高焓E⇌E‘转变有关。在这项研究中，我们测量了在不同的磷酸盐、醋酸盐和氢离子浓度组合下，酶-NADPH结合的热变和质子转移。在醋酸盐-NADPH和H+-磷酸盐对中观察到正相互作用，而在H+-NADPH、磷酸盐-NADPH、醋酸盐-磷酸盐和H+-醋酸酯对中观察到负相互作用。我们提出了一个通用模型来解释所有这些影响。这个模型结合了一个新定义的辅酶结合子。观察到的现象是根据特定阴离子结合位点在调节pK8.5酶群电离的酶上的负载程度来解释的。在这个位置上，质子与两个磷酸基团协同共享。此外，我们得出结论，这种与酶结合的合作三分子构成了在该酶的配体结合反应中观察到的高焓两态转变的变构驱动力。
It is known that the binding of the reduced coenzyme (NADPH) to bovine liver glutamate dehydrogenase is controlled by the presence of phosphate, acetate, and other anions as well as the pH of the medium. These effecters mediate this binding by lowering the pK(8.5) of an ionizable group on the enzyme, and this pKshift is linked to a high enthalpy E ⇌ E′ transition in the protein. In this study, we have measured enthalpy changes and proton transfer for enzyme-NADPH binding under a variety of combinations of phosphate, acetate, and hydrogen ion concentrations. Positive interactions are observed in the pairs acetate-NADPH and H+-phosphate, and negative interactions are seen in the pairs H+-NADPH, phosphate-NADPH, acetate-phosphate, and H+-acetate. We present a general model to account for all of these effects. This model incorporates a newly defined coenzyme binding subsite. The observed phenomena are interpreted in terms of the extent of loading of the specific anion-binding site on the enzyme that regulates the ionization of an enzyme group of pK8.5. A proton is cooperatively shared with two phosphate groups at this site. Furthermore, we conclude that this cooperative trimolecular binding to the enzyme constitutes an allosteric driving force for the high enthalpy two-state transition observed in the ligand binding reactions of this enzyme.