Tsunematsu H, Nishikawa H, Berliner L J
J Biochem. 1984 Aug;96(2):349-55. doi: 10.1093/oxfordjournals.jbchem.a134844.
Active site Ser-195-fluorine-labeled derivatives of alpha-chymotrypsin were prepared from a series of N-(trifluoromethylphenyl)-fluorosulfonylphenyl carboxamides whose synthesis is described. The six new 19F spin labels varied in the position of the -CF3 substituent (o-, m-, and p-) and the fluorosulfonyl substituent (m- or p-). The chemical shifts of these covalently bound analogs of "tosyl-chymotrypsin" were each uniquely sensitive to their environment in the catalytic center as evidenced by differences in resonance line position for each label. Upon titrating these derivatives with the reversible competitive inhibitor, indole, a downfield shift was observed (with all but one label), which could be fit in each case to an apparent dissociation constant for indole binding. Indole binding to the p-sulfonyl derivatives was essentially unaltered from that for the native enzyme, while the m-sulfonyl derivatives required some additional free energy of binding to saturate the enzyme. The results are consistent with a partial embedding of the phenylsulfonyl moiety in the aromatic specificity pocket.
从一系列已描述合成方法的N-（三氟甲基苯基）-氟磺酰基苯基羧酰胺制备了α-胰凝乳蛋白酶的活性位点丝氨酸-195氟标记衍生物。这六种新的19F自旋标记物在-CF3取代基（邻位、间位和对位）和氟磺酰基取代基（间位或对位）的位置上有所不同。这些“甲苯磺酰基-胰凝乳蛋白酶”共价结合类似物的化学位移对其在催化中心的环境都具有独特的敏感性，每个标记物的共振线位置差异证明了这一点。在用可逆竞争性抑制剂吲哚滴定这些衍生物时，观察到了向下场的位移（除一个标记物外均如此），在每种情况下，该位移都可以拟合为吲哚结合的表观解离常数。吲哚与对磺酰基衍生物的结合与天然酶基本相同，而间磺酰基衍生物需要一些额外的结合自由能来使酶饱和。结果与苯基磺酰基部分部分嵌入芳香族特异性口袋一致。
