Hydrogen bonding to active-site histidine in peptidyl boronic acid inhibitor complexes of chymotrypsin and subtilisin: Proton magnetic resonance assignments and H D fractionation
Hydrogen bonding to active-site histidine in peptidyl boronic acid inhibitor complexes of chymotrypsin and subtilisin: Proton magnetic resonance assignments and H D fractionation
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DOI：
10.1021/ja990180g
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发表时间：
1999-05-19
影响因子：
15
通讯作者：
Jordan, F
中科院分区：
文献类型：
--
作者：
Bao, DH;Huskey, WP;Jordan, F
对于MeOSuc-Ala-Ala-Pro-boroPhe（BoroPhe）与胰凝乳蛋白酶的复合物的活性中心His 57的（NH）-H-delta 1（16.9 ppm）和（NH）-H-delta 2（16.1 ppm）以及对于未复合的胰凝乳蛋白酶中的His 57的（CH）-H-delta 1质子（在低pH下9.2 ppm和在高pH下8.5 ppm），建立了H-1 NMR化学位移归属。对（CH）-H-NH 1的分配纠正了以前的分配，并揭示了这个碳结合质子的不寻常环境。相对的NH分配是从以前发现的α-裂解蛋白酶复合物与硼酸盐抑制剂的NH分配的顺序颠倒。同位素分馏因子（H/D）测定使用H-1 NMR的氢键的活性位点组氨酸在BoroPhe复合物与胰凝乳蛋白酶和枯草杆菌蛋白酶E，和未复合的胰凝乳蛋白酶。对于未复合的胰凝乳蛋白酶的（NH）-H-δ 1质子，精确到约+/-0.1的测量分馏因子为0.82（pH 10）和0.64（pH 3）。在pH 6.5的BoroPhe存在下，胰凝乳蛋白酶-抑制剂复合物的（NH）-H-δ 1分级因子为0.65，枯草杆菌蛋白酶-抑制剂复合物的分级因子为0.53。（NH）-H-Phe 2分级因子的测量值为1.05（pH 10下的未复合胰凝乳蛋白酶）、0.93（pH 6.5下的BoroPhe-胰凝乳蛋白酶）和0.76（pH 6.5下的BoroPhe-枯草杆菌蛋白酶）。模型计算的同位素分馏因子和实验测定的抑制常数被用于分馏因子结果的分析。
H-1 NMR chemical shift assignments were established for (NH)-H-delta 1 (16.9 ppm) and (NH)-H-epsilon 2 (16.1 ppm) of the active-center His57 for the complex of MeOSuc-Ala-Ala-Pro-boroPhe (BoroPhe) with chymotrypsin and for the (CH)-H-epsilon 1 proton (9.2 ppm at low pH and 8.5 ppm at high pH) of His57 in uncomplexed chymotrypsin. The assignment for (CH)-H-epsilon 1 corrects previous assignments and reveals an unusual environment of this carbon-bound proton. The relative NH assignments are reversed from the order of NH assignments previously found for alpha-lytic protease complexes with boronate inhibitors. Isotopic fractionation factors (H/D) were determined using H-1 NMR for hydrogen bonds to the active site histidine in BoroPhe complexes with chymotrypsin and subtilisin E, and for uncomplexed chymotrypsin. Measured fractionation factors accurate to about +/-0.1 were 0.82 (pH 10) and 0.64 (pH 3) for the (NH)-H-delta 1 proton of uncomplexed chymotrypsin. In the presence of BoroPhe at pH 6.5, the (NH)-H-delta 1 fractionation factors were 0.65 for the chymotrypsin-inhibitor complex, and 0.53 for the subtilisin-inhibitor complex. Measurements for the (NH)-H-epsilon 2 fractionation factor were 1.05 (uncomplexed chymotrypsin at pH 10), 0.93 (BoroPhe-chymotrypsin at pH 6.5), and 0.76 (BoroPhe-subtilisin at pH 6.5). Both model calculations of isotopic fractionation factors and experimentally determined inhibition constants were used in the analysis of the fractionation-factor results.