Bah Alaji, Garvey Laura C, Ge Jingping, Di Cera Enrico
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
J Biol Chem. 2006 Dec 29;281(52):40049-56. doi: 10.1074/jbc.M608600200. Epub 2006 Oct 30.
The kinetic mechanism of Na(+) binding to thrombin was resolved by stopped-flow measurements of intrinsic fluorescence. Na(+) binds to thrombin in a two-step mechanism with a rapid phase occurring within the dead time of the spectrometer (<0.5 ms) followed by a single-exponential slow phase whose k(obs) decreases hyperbolically with increasing [Na(+)]. The rapid phase is due to Na(+) binding to the enzyme E to generate the E:Na(+) form. The slow phase is due to the interconversion between E() and E, where E() is a form that cannot bind Na(+). Temperature studies in the range from 5 to 35 degrees C show significant enthalpy, entropy, and heat capacity changes associated with both Na(+) binding and the E to E() transition. As a result, under conditions of physiologic temperature and salt concentrations, the E() form is negligibly populated (<1%) and thrombin is almost equally partitioned between the E (40%) and E:Na(+) (60%) forms. Single-site Phe mutations of all nine Trp residues of thrombin enabled assignment of the fluorescence changes induced by Na(+) binding mainly to Trp-141 and Trp-215, and to a lesser extent to Trp-148, Trp-207, and Trp-237. However, the fast phase of fluorescence increase is influenced to different extents by all Trp residues. The distribution of these residues over the entire thrombin surface demonstrates that Na(+) binding induces long-range effects on the structure of the enzyme as a whole, contrary to the conclusions drawn from recent structural studies. These findings elucidate the mechanism of Na(+) binding to thrombin and are relevant to other clotting factors and enzymes allosterically activated by monovalent cations.
通过对固有荧光的停流测量解析了Na⁺与凝血酶结合的动力学机制。Na⁺以两步机制与凝血酶结合，快速相发生在光谱仪的死时间内（<0.5毫秒），随后是单指数慢相，其k(obs)随[Na⁺]增加呈双曲线下降。快速相是由于Na⁺与酶E结合生成E:Na⁺形式。慢相是由于E()和E之间的相互转化，其中E()是一种不能结合Na⁺的形式。在5至35摄氏度范围内的温度研究表明，与Na⁺结合以及E向E()转变相关的焓、熵和热容都有显著变化。因此，在生理温度和盐浓度条件下，E()形式的占比可忽略不计（<1%），凝血酶几乎平均分配在E（40%）和E:Na⁺（60%）形式之间。凝血酶所有九个色氨酸残基的单点苯丙氨酸突变使得能够将Na⁺结合诱导的荧光变化主要归因于色氨酸-141和色氨酸-215，在较小程度上归因于色氨酸-148、色氨酸-207和色氨酸-237。然而，荧光增加的快速相受到所有色氨酸残基不同程度的影响。这些残基在整个凝血酶表面的分布表明，与最近结构研究得出的结论相反，Na⁺结合对整个酶的结构产生了远程影响。这些发现阐明了Na⁺与凝血酶结合的机制，并且与其他凝血因子以及由单价阳离子变构激活的酶相关。
