Goux W J, Teherani J, Sherry A D
Biophys Chem. 1984 Jun;19(4):363-73. doi: 10.1016/0301-4622(84)87019-2.
Exchange rates were calculated as a function of pH from line widths of methylamine resonances in 13C-NMR spectra of N epsilon,N epsilon,N alpha,N alpha-[13C]tetramethyllysine (TML) and N epsilon,N epsilon,N alpha,N alpha-tetramethyllysine methyl ester (TMLME). The pH dependence of the dimethyl alpha-amine exchange rate could be adequately described by assuming base-catalyzed chemical exchange between two diastereotopic methyl populations related by nitrogen inversion. Deprotonation of the alpha-amine was assumed to occur by proton transfer to (1) OH-, (2) water, (3) a deprotonated amine or (4) RCO2-. Microscopic rate constants characterizing each of these transfer processes (k1, k2, k3 and k4, respectively) were determined by fitting the rates calculated from line width analysis to a steady-state kinetic model. Using this procedure it was determined that for both TML and TMLME k2 approximately equal to 1-10 M-1 s-1, k3 approximately equal to 10(6) M-1 s-1 and ki, the rate constant for nitrogen inversion was about 10(8)-10(9) s-1. Upper limits of 10(12) and 10(3) M-1 s-1 could be determined for k1 and k4, respectively. A similar kinetic analysis was used to explain pH-dependent line-broadening effects observed for the N-terminal dimethylalanyl resonance in 13C-NMR spectra of concanavalin A, reductively methylated using 90% [13C]formaldehyde. From exchange data below pH 4 it could be determined that amine inversion was limited by the proton transfer rate to the solvent, with a rate constant estimated at 20 M-1 s-1. Above pH 4, exchange was limited by proton transfer to other titrating groups in the protein structure. Based upon their proximity, the carboxylate side chains of Asp-2 and Asp-218 appear to be likely candidates. The apparent first-order microscopic rate constant characterizing proton transfer to these groups was estimated to be about 1 X 10(4) s-1. Rate constants characterizing nitrogen inversion (ki), proton transfer to OH- (k1) and proton transfer to the solvent (k2) were estimated to be of the same order of magnitude as those determined for the model compounds. On the basis of our results, it is proposed that chemical exchange processes associated with base-catalyzed nitrogen inversion may contribute to 15N or 13C spin-lattice relaxation times in reductively methylated peptides or proteins.
通过测定Nε,Nε,Nα,Nα-[13C]四甲基赖氨酸（TML）和Nε,Nε,Nα,Nα-四甲基赖氨酸甲酯（TMLME）的13C-NMR谱中甲基胺共振峰的线宽，计算出交换速率与pH的函数关系。通过假设氮翻转相关的两个非对映异位甲基基团之间的碱催化化学交换，可以充分描述二甲基α-胺交换速率对pH的依赖性。假设α-胺的去质子化是通过质子转移至（1）OH-、（2）水、（3）去质子化的胺或（4）RCO2-而发生的。通过将线宽分析计算得到的速率拟合到稳态动力学模型，确定了表征这些转移过程的微观速率常数（分别为k1、k2、k3和k4）。使用该方法确定，对于TML和TMLME，k2约等于1 - 10 M-1 s-1，k3约等于10(6) M-1 s-1，氮翻转的速率常数ki约为10(8)-10(9) s-1。k1和k4的上限分别可以确定为10(12)和10(3) M-1 s-1。类似的动力学分析用于解释伴刀豆球蛋白A的13C-NMR谱中N端二甲基丙氨酰共振峰观察到的pH依赖性线宽效应，该伴刀豆球蛋白A使用90% [13C]甲醛进行了还原甲基化。从pH 4以下的交换数据可以确定，胺翻转受质子转移至溶剂的速率限制，估计速率常数为20 M-1 s-1。在pH 4以上，交换受质子转移至蛋白质结构中其他滴定基团的限制。基于它们的接近程度，Asp-2和Asp-218的羧酸盐侧链似乎是可能的候选基团。表征质子转移至这些基团的表观一级微观速率常数估计约为1×10(4) s-1。表征氮翻转（ki）、质子转移至OH-（k1）和质子转移至溶剂（k2）的速率常数估计与模型化合物测定的速率常数处于同一数量级。基于我们的结果，提出与碱催化氮翻转相关的化学交换过程可能对还原甲基化肽或蛋白质中的15N或13C自旋晶格弛豫时间有贡献。
