Tóth Kamil, Sedlák Erik, Sprinzl Mathias, Zoldák Gabriel
Department of Biochemistry, Faculty of Sciences P. J. Safárik University, Kosice, Slovakia.
Biochim Biophys Acta. 2008 May;1784(5):789-95. doi: 10.1016/j.bbapap.2008.01.022. Epub 2008 Feb 20.
Recently, we have shown that anions of Hofmeister series affect the enzyme activity through modulation of flexibility of its active site. The enzyme activity vs. anion position in Hofmeister series showed an unusual bell-shaped dependence. In the present work, six monovalent cations (Na(+), Gdm(+), NH(4)(+), Li(+), K(+) and Cs(+)) of Hofmeister series with chloride as a counterion have been studied in relation to activity and stability of flavoprotein NADH oxidase from Thermus thermophilus (NOX). With the exception of strongly chaotropic guanidinium cation, cations are significantly less effective in promoting the Hofmeister effect than anions mainly due to repulsive interactions of positive charges around the active site. Thermal denaturations of NOX reveal unfavorable electrostatic interaction at the protein surface that may be shielded to different extent by salts. Michaelis-Menten constants for NADH, accessibility of the active site as reflected by Stern-Volmer constants and activity of NOX at high cation concentrations (1-2 M) show bell-shaped dependences on cation position in Hofmeister series. Our analysis indicates that in the presence of kosmotropic cations the enzyme is more stable and possibly more rigid than in the presence of chaotropic cations. Molecular dynamic (MD) simulations of NOX showed that active site switches between open and closed conformations [J. Hritz, G. Zoldak, E. Sedlak, Cofactor assisted gating mechanism in the active site of NADH oxidase from Thermus thermophilus, Proteins 64 (2006) 465-476]. Enzyme activity, as well as substrate binding, can be regulated by the salt mediated perturbation of the balance between open and closed forms. We propose that compensating effect of accessibility and flexibility of the enzyme active site leads to bell-shaped dependence of the investigated parameters.
最近，我们已经表明，霍夫迈斯特序列的阴离子通过调节其活性位点的柔韧性来影响酶活性。酶活性与霍夫迈斯特序列中阴离子位置的关系呈现出一种不寻常的钟形依赖性。在本工作中，研究了以氯离子为抗衡离子的霍夫迈斯特序列中的六种单价阳离子（Na⁺、Gdm⁺、NH₄⁺、Li⁺、K⁺和Cs⁺）对嗜热栖热菌黄素蛋白NADH氧化酶（NOX）活性和稳定性的影响。除了具有强离液序列高的胍阳离子外，阳离子在促进霍夫迈斯特效应方面的效果明显低于阴离子，这主要是由于活性位点周围正电荷的排斥相互作用。NOX的热变性揭示了蛋白质表面存在不利的静电相互作用，而盐可以不同程度地屏蔽这种相互作用。NADH的米氏常数、由斯特恩-沃尔默常数反映的活性位点可及性以及高阳离子浓度（1 - 2 M）下NOX的活性对霍夫迈斯特序列中阳离子位置呈现出钟形依赖性。我们的分析表明，在存在促溶剂化阳离子的情况下，酶比存在离液序列高的阳离子时更稳定，可能也更刚性。NOX的分子动力学（MD）模拟表明，活性位点在开放和闭合构象之间切换[J. Hritz, G. Zoldak, E. Sedlak, Cofactor assisted gating mechanism in the active site of NADH oxidase from Thermus thermophilus, Proteins 64 (2006) 465 - 476]。酶活性以及底物结合可以通过盐介导的开放和闭合形式之间平衡的扰动来调节。我们提出，酶活性位点可及性和柔韧性的补偿效应导致了所研究参数的钟形依赖性。
