Tang Aaron W, Kong Xianqi, Terskikh Victor, Wu Gang
Department of Chemistry, Queen's University , 90 Bader Lane, Kingston, Ontario K7L 3N6, Canada.
Department of Chemistry, University of Ottawa , Ottawa, Ontario K1N 6N5, Canada.
J Phys Chem B. 2016 Nov 3;120(43):11142-11150. doi: 10.1021/acs.jpcb.6b08798. Epub 2016 Oct 21.
We report preparation, trapping, and solid-state O NMR characterization of three unstable acyl-enzyme intermediates (≈ 26 kDa): p-N,N-dimethylamino-[O]benzoyl-chymotrypsin, trans-o-methoxy-[O]cinnamoyl-chymotrypsin, and trans-p-methoxy-[O]cinnamoyl-chymotrypsin. We show that both the O chemical shifts and nuclear quadrupolar parameters obtained for these acyl-enzyme intermediates in the solid state are correlated with their deacylation rate constants measured in aqueous solution. With the aid of quantum mechanical calculations, the experimental O NMR parameters were interpreted as to reflect the hydrogen bonding interactions between the carbonyl (C═O) functional group of the acyl moiety and the two NH groups from the protein backbone (Ser195 and Gly193) in the oxyanion hole, a general feature of all serine proteases. Our results further suggest that the O chemical shift and quadrupole coupling constant display distinctly different sensitivities toward different aspects of hydrogen bonding, such as hydrogen bond distance and direction. This work demonstrates the utility of O as a useful nuclear probe in NMR studies of enzymes.
我们报告了三种不稳定的酰基酶中间体（约26 kDa）的制备、捕获及固态¹⁷O NMR表征：对-N，N-二甲基氨基-[¹⁷O]苯甲酰基胰凝乳蛋白酶、反式邻甲氧基-[¹⁷O]肉桂酰基胰凝乳蛋白酶和反式对甲氧基-[¹⁷O]肉桂酰基胰凝乳蛋白酶。我们表明，这些酰基酶中间体在固态下获得的¹⁷O化学位移和核四极参数与其在水溶液中测得的脱酰基速率常数相关。借助量子力学计算，对实验性¹⁷O NMR参数进行了解释，以反映酰基部分的羰基（C═O）官能团与氧负离子洞中来自蛋白质主链（Ser195和Gly193）的两个NH基团之间的氢键相互作用，这是所有丝氨酸蛋白酶的一个普遍特征。我们的结果进一步表明，¹⁷O化学位移和四极耦合常数对氢键的不同方面（如氢键距离和方向）表现出明显不同的敏感性。这项工作证明了¹⁷O作为酶NMR研究中一种有用的核探针的实用性。
