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首页> 外文期刊>Biochemistry >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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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

机译：His 57的碱性与过渡态类似物结合，底物反应性以及胰凝乳蛋白酶中低阻隔氢键强度的相关性

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摘要

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. [References: 32]
机译：低壁垒氢键（LBHB）diad His 57-Asp 102中His57-N-ε2的碱性和胰凝乳蛋白酶与肽基三氟甲基酮的四面体，半缩合物中LBHB质子的H-1 NMR化学位移（肽基-TFK）已被研究。在5摄氏度，N-Ac-Gly-DL-Phe-CF3，pK（a）= 11.1和delta（LBHB）= 18.7 ppm的条件下，使用各种肽基-TFK可获得以下结果。 N-Ac-L-Val-DL-Phe-CF3，pK（a）= 11.8和δ（LBHB）= 18.9 ppm; N-Ac-L-Leu-DL-Val-CF3，pK（a）= 10.3，δ（LBHB）= 18.9 ppm;和N-Ac-L-Leu-DL-萘基CF3，pK（a）= 10.9和delta（LBHB）= 19.0 ppm在P-1处有Phe且N-Ac，N-Ac的肽基TFK的结果在P-2位的-Gly，N-Ac-L-Val和N-Ac-L-Leu与相应的肽基甲基酯的抑制常数Ki和k（cat）/ Km的文献值充分相关。 log Ki对His57-N-ε2的表观pK（a）的曲线显示出-0.77的斜率，而肽基甲基酯的log k（cat）/ Km对log与His 57的pK（a）的曲线显示。相应的肽基-TFK配合物中的-N-ε2的斜率是0.68，肽基甲基酯底物的pK（a）对delta（LBHB）的图的斜率是3.7，对数k（cat）/ Km的图斜率。相应的肽基-TFK-胰凝乳蛋白酶复合物的相对于delta（LBHB）的相对值是2.7，log Ki与delta（LBHB）的对数图显示了-3.0的斜率，这些图表明His 57的pK（a）和底物反应性为与低势垒氢键强度的增加有关。胰凝乳蛋白酶-N-Ac-L-Leu-DL-Phe-CF3复合物的His57-N-ε2的表观pK（a）在25摄氏度时为10.6，而在5摄氏度时为12.0 [Cassidy， CS，Lin，JL和Frey，PA（1997）Biochemistry 34，4576-4584]，明显的差异很可能是由于肽57-TFK络合物中His 57的协同电离中的温度依赖性所致。抑制游离肽基-TFK的解离，水合和离子化，Ile 16的离子化以及构象变化。 [参考：32]

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