Dupaix A, Bechet J J, Yon J, Merlin J C, Delhaye M, Hill M
Proc Natl Acad Sci U S A. 1975 Nov;72(11):4223-7. doi: 10.1073/pnas.72.11.4223.
Raman spectroscopy was used to study the interactions between bovine trypsin and a competitive inhibitor. For this purpose, a chromophoric substrate analogue, 4-amidino-4'-dimethylamine azobenzene, was synthesized. This compound competitively inhibits the enzyme with a 1:1 stoichiometry and an inhibition constant Ki of 2.3 muM at pH 6.08 and 15 degrees. Resonance Raman spectra in aqueous solution of free or enzyme-bound inhibitor were analyzed. The main spectral changes observed upon enzyme-inhibitor complex formation were changes in the relative intensities of four bands (1171, 1206, 1315, 1608 cm-1) while no large frequency shifts occurred. The binding of the inhibitor molecule to the enzyme did not induce a twisting of the phenyl groups around the N=N bond. Some modifications of the band widths are interpreted in terms of a restriction of rotational motions in the inhibitor molecule. The possible involvement of specific interactions between trypsin and the benzamidinium ion part of the inhibitor molecule is discussed.
采用拉曼光谱法研究牛胰蛋白酶与一种竞争性抑制剂之间的相互作用。为此，合成了一种发色底物类似物，即4-脒基-4'-二甲胺基偶氮苯。该化合物以1:1的化学计量比竞争性抑制该酶，在pH 6.08和15℃条件下抑制常数Ki为2.3 μM。分析了游离或与酶结合的抑制剂在水溶液中的共振拉曼光谱。酶-抑制剂复合物形成时观察到的主要光谱变化是四条谱带（1171、1206、1315、1608 cm-1）相对强度的变化，而没有发生大的频率偏移。抑制剂分子与酶的结合并未诱导苯基围绕N=N键发生扭转。根据抑制剂分子中旋转运动的受限情况对谱带宽度的一些变化进行了解释。讨论了胰蛋白酶与抑制剂分子中苯甲脒离子部分之间特定相互作用的可能参与情况。
