Correlations of the basicity of His 57 with transition state analogue binding, substrate reactivity, and the strength of the low-barrier hydrogen bond in chymotrypsin
Correlations of the basicity of His 57 with transition state analogue binding, substrate reactivity, and the strength of the low-barrier hydrogen bond in chymotrypsin
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DOI：
10.1021/bi980278s
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发表时间：
1998-08-25
期刊：
影响因子：
2.9
通讯作者：
Frey, PA
中科院分区：
文献类型：
--
作者：
Lin, J;Cassidy, CS;Frey, PA
本文研究了h57 - n- epsilon 2在低势垒氢键（LBHB）和h57 - asp 102中的碱度，以及LBHB质子在凝乳胰蛋白酶与肽基三氟甲基酮（肽基- tfks）的四面体半晶配合物中的H-1 NMR化学位移。在5℃，n - ac - gly - dl - ph - cf3, pK(a) = 11.1, δ (LBHB) = 18.7 ppm条件下，不同的肽基- tfks得到如下结果；n - ac - l- val - dl - ph - cf3, pK(a) = 11.8, δ (LBHB) = 18.9 ppm；N-Ac-L-Leu-DL-Val-CF3, pK(a) = 10.3, δ (LBHB) = 18.9 ppm；P-1位的Phe和P-2位的N-Ac、N-Ac- gly、N-Ac- l- val和N-Ac- l- leu的肽基tfks的抑制常数k -i和k(cat)/ k -m与文献中相应肽基甲酯的抑制常数k(i)和k(cat)/ k -m具有良好的相关性。log k -i与His 57-N-epsilon 2表观pK(a)的斜率为-0.77，对应肽基- tfk配合物中His 57-N-epsilon 2表观pK(a)的斜率为log k(cat)/ k -m，对应肽基- tfk -糜蛋白酶配合物中His 57-N-epsilon 2表观pK(a)的斜率为0.68,pK(a)与delta（LBHB）的斜率为3.7,logk (cat)/ k -m与delta（LBHB）的斜率为2.7。log K-i - δ (LBHB)曲线斜率为-3.0，表明His 57的pK(A)和底物反应性与低势垒氢键强度的增加有关。明显pK (a)他的57-N-epsilon chymotrypsin-N-Ac-L-Leu-DL-Phe-CF3复杂2是10.6在25摄氏度,而它是12.0在5摄氏度(C . S。卡西迪,林,j·l·弗雷,p . a(1997)生物化学34岁,4576 - 4584],明显的差异可能是由于温度依赖的合作电离57 peptidyl-TFK复合物,这似乎是离解耦合剂,水化自由peptidyl-TFK电离,电离Ile 16日还有构象变化。
The basicity of His 57-N-epsilon 2 within the low-barrier hydrogen-bonded (LBHB) diad His 57-Asp 102 and the H-1 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, pK(a) = 11.1 and delta(LBHB) = 18.7 ppm; N-Ac-L-Val-DL-Phe-CF3, pK(a) = 11.8 and delta(LBHB) = 18.9 ppm; N-Ac-L-Leu-DL-Val-CF3, pK(a) = 10.3 and delta(LBHB) = 18.9 ppm; and N-Ac-L-Leu-DL-naphthyl-CF3, pK(a) = 10.9 and delta(LBHB) = 19.0 ppm Results for peptidyl-TFKs with Phe in the P-1 position and N-Ac, N-Ac-Gly, N-Ac-L-Val, and N-Ac-L-Leu in the P-2 position were well correlated with literature values for inhibition constants K-i and k(cat)/K-m for the corresponding peptidyl methyl esters. The plot of log K-i versus the apparent pK(a) of His 57-N-epsilon 2 displayed a slope of -0.77, and that of log k(cat)/K-m for peptidyl methyl esters versus the pK(a) of His 57-N-epsilon 2 in corresponding peptidyl-TFK complexes gave a slope of 0.68, The slope of a plot of pK(a) versus delta(LBHB) was 3.7, and that of log k(cat)/K-m for peptidyl methyl ester substrates versus delta(LBHB) for the corresponding peptidyl-TFK-chymotrypsin complexes was 2.7, A plot of log K-i versus delta(LBHB) displayed a slope of -3.0, These plots indicated that the pK(a) of His 57 and substrate reactivity were correlated with increasing strength of the low-barrier hydrogen bond. The apparent pK(a) of His 57-N-epsilon 2 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 34, 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.