COMPLEX OF ALPHA-CHYMOTRYPSIN AND N-ACETYL-L-LEUCYL-L-PHENYLALANYL TRIFLUOROMETHYL KETONE - STRUCTURAL STUDIES WITH NMR-SPECTROSCOPY
COMPLEX OF ALPHA-CHYMOTRYPSIN AND N-ACETYL-L-LEUCYL-L-PHENYLALANYL TRIFLUOROMETHYL KETONE - STRUCTURAL STUDIES WITH NMR-SPECTROSCOPY
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DOI：
10.1021/bi00398a011
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发表时间：
1987-12-01
期刊：
影响因子：
2.9
通讯作者：
ABELES, RH
中科院分区：
文献类型：
--
作者：
LIANG, TC;ABELES, RH
合成了二肽基三氟甲基酮，即N-乙酰基-L-亮氨酰-L-[1- 13 C]苯丙氨酰三氟甲基酮。该化合物抑制糜蛋白酶，Ki = 1.2 μ M [Imperiali B.，和Abeles，R. H.（1986）Biochemistry 25，3760-3767]。该抑制剂与α-葡萄糖形成的复合物胰凝乳蛋白酶用1H、13 C和19 F NMR光谱检查以确定其在溶液中的结构。三氟酮的酮基作为离子化的半缩酮基团存在，这是从其13 C化学位移与模型半缩酮的化学位移的比较中推断出的。复合物中半缩酮羟基的pKa约为4.9，比模型半缩酮的pKa低约4.2个单位。这一观察结果提供了丝氨酸蛋白酶能够稳定四面体加合物的含氧阴离子的直接证据。证据也提出了一个Asp-His H键和质子化的咪唑基团的His-57的四面体加合物的存在下。His-57的pKa大于10。这一观察结果直接表明，在含有四面体加合物的复合物中，His-57的pKa升高。
A dipeptidyl trifluoromethyl ketone, N-acetyl-L-leucyl-L-[1-13C]phenylalanyl trifluoromethyl ketone, was synthesized. This compound inhibits chymotrypsin with Ki = 1.2 .mu.M [Imperiali B., and Abeles, R. H. (1986) Biochemistry 25, 3760-3767]. The complex formed between this inhibitor and .alpha.-chymotrypsin was examined with 1H, 13C, and 19F NMR spectroscopy to establish its structure in solution. The keto group of the trifluoro ketone is present as an ionized hemiketal group as deduced from the comparison of its 13C chemical shift with those of model hemiketals. The pKa of the hemiketal hydroxyl in the complex is approximately 4.9, which is about 4.2 units lower than the pKa of model hemiketals. This observation provides direct evidence that serine proteases are able to stabilize the oxyanions of tetrahedral adducts. Evidence is also presented for the presence of an Asp-His H bond and protonation of the imidazole group of His-57 in the tetrahedral adduct. The pKa of His-57 is higher than 10. This observation directly indicates that the pKa of His-57 is elevated in a complex containing a tetrahedral adduct.