Lin J, Cassidy C S, Frey P A
Institute for Enzyme Research, The Graduate School, College of Agricultural and Life Sciences, University of Wisconsin-Madison 53705, USA.
Biochemistry. 1998 Aug 25;37(34):11940-8. doi: 10.1021/bi980278s.
The basicity of His 57-Nepsilon2 within the low-barrier hydrogen-bonded (LBHB) diad His 57-Asp 102 and the 1H NMR chemical shift of the LBHB proton in tetrahedral, hemiketal complexes of chymotrypsin with peptidyl trifluoromethyl ketones (peptidyl-TFKs) have been studied. The following results were obtained with various peptidyl-TFKs at 5 degrees C: N-Ac-Gly-DL-Phe-CF3, pKa = 11.1 and deltaLBHB = 18.7 ppm; N-Ac-L-Val-DL-Phe-CF3, pKa = 11.8 and deltaLBHB = 18.9 ppm; N-Ac-L-Leu-DL-Val-CF3, pKa = 10.3 and deltaLBHB = 18.9 ppm; and N-Ac-L-Leu-DL-naphthyl-CF3, pKa = 10.9 and deltaLBHB = 19.0 ppm. Results for peptidyl-TFKs with Phe in the P1 position and N-Ac, N-Ac-Gly, N-Ac-L-Val, and N-Ac-L-Leu in the P2 position were well correlated with literature values for inhibition constants Ki and kcat/Km for the corresponding peptidyl methyl esters. The plot of log Ki versus the apparent pKa of His 57-Nepsilon2 displayed a slope of -0.77, and that of log kcat/Km for peptidyl methyl esters versus the pKa of His 57-Nepsilon2 in corresponding peptidyl-TFK complexes gave a slope of 0.68. The slope of a plot of pKa versus deltaLBHB was 3.7, and that of log kcat/Km for peptidyl methyl ester substrates versus deltaLBHB for the corresponding peptidyl-TFK-chymotrypsin complexes was 2.7. A plot of log Ki versus deltaLBHB displayed a slope of -3.0. These plots indicated that the pKa of His 57 and substrate reactivity were correlated with increasing strength of the low-barrier hydrogen bond. The apparent pKa of His 57-Nepsilon2 for the chymotrypsin-N-Ac-L-Leu-DL-Phe-CF3 complex is 10.6 at 25 degrees C, whereas it is 12.0 at 5 degrees C [Cassidy, C. S., Lin, J. L., and Frey, P. A. (1997) Biochemistry 36, 4576-4584]. The apparent discrepancy is likely to be due to a temperature dependence in the cooperative ionization of His 57 in peptidyl-TFK complexes, which appears to be coupled to inhibitor dissociation, hydration and ionization of free peptidyl-TFK, ionization of Ile 16, and a conformational change.
研究了在低势垒氢键（LBHB）二元组His 57 - Asp 102中His 57 - Nε2的碱性以及胰凝乳蛋白酶与肽基三氟甲基酮（肽基 - TFKs）的四面体、半缩酮络合物中LBHB质子的1H NMR化学位移。在5℃下用各种肽基 - TFKs得到以下结果：N - Ac - Gly - DL - Phe - CF3，pKa = 11.1且δLBHB = 18.7 ppm；N - Ac - L - Val - DL - Phe - CF3，pKa = 11.8且δLBHB = 18.9 ppm；N - Ac - L - Leu - DL - Val - CF3，pKa = 10.3且δLBHB = 18.9 ppm；以及N - Ac - L - Leu - DL - 萘基 - CF3，pKa = 10.9且δLBHB = 19.0 ppm。P1位为苯丙氨酸且P2位为N - Ac、N - Ac - Gly、N - Ac - L - Val和N - Ac - L - Leu的肽基 - TFKs的结果与相应肽基甲酯的抑制常数Ki和kcat/Km的文献值具有良好的相关性。log Ki对His 57 - Nε2的表观pKa作图，斜率为 - 0.77，肽基甲酯的log kcat/Km对相应肽基 - TFK络合物中His 57 - Nε2的pKa作图，斜率为0.68。pKa对δLBHB作图的斜率为3.7，肽基甲酯底物的log kcat/Km对相应肽基 - TFK - 胰凝乳蛋白酶络合物的δLBHB作图的斜率为2.7。log Ki对δLBHB作图的斜率为 - 3.0。这些作图表明His 57的pKa和底物反应性与低势垒氢键强度的增加相关。胰凝乳蛋白酶 - N - Ac - L - Leu - DL - Phe - CF3络合物中His 57 - Nε2在25℃时的表观pKa为10.6，而在5℃时为12.0 [卡西迪，C.S.，林，J.L.，和弗雷，P.A.（1997年）《生物化学》36，4576 - 4584]。这种明显的差异可能是由于肽基 - TFK络合物中His 57协同电离的温度依赖性，这似乎与抑制剂解离、游离肽基 - TFK的水合和电离、Ile 16的电离以及构象变化有关。
