Pressure dependence of amide hydrogen-deuterium exchange rates for individual sites in T4 lysozyme.
Pressure dependence of amide hydrogen-deuterium exchange rates for individual sites in T4 lysozyme.
复制标题
T4 溶菌酶中各个位点酰胺氢-氘交换率的压力依赖性。
DOI：
10.1021/bi972950b
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发表时间：
1998
期刊：
影响因子：
--
通讯作者：
Bryant,RG
中科院分区：
文献类型：
--
作者：
Hitchens,TK;Bryant,RG
我们报告测量的压力依赖性的速率常数的酰胺残基质子与溶剂氘T4溶菌酶的交换。在9个压力从0.1到200 MPa的数据进行了分析，使用一个基本的动力学模型和过渡态理论的形式主义，产生的交换过程的活化体积。使用HSQC二维（2D）NMR实验对均匀15 N标记的蛋白质完成单个酰胺位点的解析。在22 °C和pH* 7.5下，观察到的活化体积范围为2.75至−25.1 mL/mol。当校正离子产物对水的压力依赖性和模型化合物中酰胺交换反应的报告活化体积时，与溶剂或催化剂对酰胺位点的可及性相关的活化体积部分范围为-15.1至12.8 mL/mol。酰胺氢交换的活化体积与保护因子之间没有简单的相关性。在一级序列或折叠结构中非常接近的残基的激活体积可能会有很大的不同。活化体积和残基所在的二级结构单元之间没有无关紧要的相关性，并且显然结构上偶联的残基的活化体积可能非常不同。在这些条件下获得的活化体积的适度大小与牛胰蛋白酶抑制剂在60 ℃和pH* 8的更极端条件下报道的大值形成对比，其中主要的解折叠事件或结构重排可能主导机制[瓦格纳，G.（1983）Q. Rev. Biopolymer.16，1 - 57]。
We report measurements of the pressure dependence of rate constants for the exchange of amide residue protons with solvent deuterium for T4 lysozyme. Data obtained at nine pressures from 0.1 to 200 MPa are analyzed using an elementary kinetic model and the formalism of transition state theory which yield activation volumes for the exchange process. Resolution of individual amide sites was accomplished using the HSQC two-dimensional (2D) NMR experiment on uniformly15N-labeled protein. The observed activation volumes span the range from 2.75 to −25.1 mL/mol at 22 °C and pH* 7.5. When corrected for the pressure dependence of the ionic product for water and for the reported activation volume for the amide exchange reaction in model compounds, the portion of the activation volume associated with the accessibility of the solvent or catalyst to the amide sites ranges from −15.1 to 12.8 mL/mol. There is no simple correlation between the activation volumes and the protection factors for amide hydrogen exchange. The activation volumes for residues in close proximity in either the primary sequence or the folded structure may differ considerably. There is no trivial correlation between the activation volume and the secondary structural unit in which a residue is located, and activation volumes for residues that are apparently structurally coupled may be very different. The modest sizes of the activation volumes obtained under these conditions are in contrast to large values reported for bovine pancreatic trypsin inhibitor at more extreme conditions of 60 °C and pH* 8 where major unfolding events or structural rearrangements may dominate the mechanism [Wagner, G. (1983)Q. Rev. Biophys.16, 1−57].