Bismuto Ettore, Nucci Roberto, Febbraio Ferdinando, Tanfani Fabio, Gentile Fabrizio, Briante Raffaella, Scirè Andrea, Bertoli Enrico, Amodeo Pietro
Dipartimento di Biochimica e Biofisica, Seconda Università di Napoli, Via Costantinopoli 16, 80138, Napoli, Italy.
Eur Biophys J. 2004 Feb;33(1):38-49. doi: 10.1007/s00249-003-0350-7. Epub 2003 Oct 15.
The perturbation induced by mono- and divalent cations on the thermophilicity and thermostability of Solfolobus solfataricus beta-glycosidase, a hyperthermophilic tetrameric enzyme, has been investigated by spectroscopic and computational simulation methods to ascertain the Hofmeister effects on two strategic protein regions identified previously. Specifically, (1). an extra segment (83-124), present only in the sequence of hyperthermophilic glycosidases and recognized as an important thermostability determinant for the enzyme structure; and (2). a restricted area of the subunit interface responsible for the quaternary structure maintenance. Mono- and divalent cations inhibit to a different extent the beta-glycosidase activity, whose kinetic constants show an apparent competitive inhibition of the catalytic process that reflects the Hofmeister order. The thermostability is also affected by the nature and charge of the cations, reaching maximal effects for the case of Mg(2+). Fourier transform infrared spectroscopy has revealed very small changes in the protein secondary structure in the presence of the investigated cations at 20 degrees C, while large effects on the protein melting temperatures are observed. Computational analysis of the enzyme structure has identified negative patches on the accessible surface of the two identified regions. Following the Hofmeister series, cations weaken the existing electrostatic network that links the extra segment to the remaining protein matrix. In particular, the perturbing action of cations could involve the ionic pair interactions E107-R245 and E109-R185, thus leading to a local destructuring of the extra segment as a possible starting event for thermal destabilization. A detailed investigation of the electrostatic network at the A-C intermolecular interface of Sbetagly after energy minimization suggests that cations could cause a strong attenuation of the ion pair interactions E474-K72 and D473-R402, with consequent partial dissociation of the tetrameric structure.
通过光谱学和计算模拟方法，研究了单价和二价阳离子对嗜热栖热菌β-糖苷酶（一种嗜热四聚体酶）的嗜热性和热稳定性的影响，以确定霍夫迈斯特效应在先前确定的两个关键蛋白质区域上的作用。具体而言，（1）. 一个仅存在于嗜热糖苷酶序列中的额外片段（83 - 124），被认为是该酶结构重要的热稳定性决定因素；（2）. 亚基界面中负责维持四级结构的受限区域。单价和二价阳离子对β-糖苷酶活性有不同程度的抑制，其动力学常数显示出对催化过程的明显竞争性抑制，这反映了霍夫迈斯特序列。阳离子的性质和电荷也会影响热稳定性，Mg(2+)的情况下达到最大效果。傅里叶变换红外光谱显示，在20℃存在所研究的阳离子时，蛋白质二级结构变化非常小，而对蛋白质解链温度有很大影响。对酶结构的计算分析确定了两个已识别区域可及表面上的负电荷区域。按照霍夫迈斯特序列，阳离子会削弱将额外片段与其余蛋白质基质连接起来的现有静电网络。特别是，阳离子的扰动作用可能涉及离子对相互作用E107 - R245和E109 - R185，从而导致额外片段的局部解构，这可能是热不稳定的起始事件。对能量最小化后的Sbetagly的A - C分子间界面处的静电网络进行详细研究表明，阳离子可能会导致离子对相互作用E474 - K72和D473 - R402的强烈减弱，从而导致四聚体结构部分解离。
