HUMAN AND MURINE CYTOTOXIC LYMPHOCYTE-T SERINE PROTEASES - SUBSITE MAPPING WITH PEPTIDE THIOESTER SUBSTRATES AND INHIBITION OF ENZYME-ACTIVITY AND CYTOLYSIS BY ISOCOUMARINS
HUMAN AND MURINE CYTOTOXIC LYMPHOCYTE-T SERINE PROTEASES - SUBSITE MAPPING WITH PEPTIDE THIOESTER SUBSTRATES AND INHIBITION OF ENZYME-ACTIVITY AND CYTOLYSIS BY ISOCOUMARINS
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DOI：
10.1021/bi00222a027
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发表时间：
1991-02-26
期刊：
影响因子：
2.9
通讯作者：
POWERS, JC
中科院分区：
文献类型：
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作者：
ODAKE, S;KAM, CM;POWERS, JC
用肽硫酯底物、肽氯甲基酮和异香豆素抑制剂研究了从细胞毒性T淋巴细胞（CTL）分离的人Q 31颗粒酶A、小鼠颗粒酶（A、B、C、D、E和F）和人颗粒酶（A、B和3）的活性部位结构。 人Q31、鼠和人颗粒酶A非常有效地水解含Arg或Lys的硫酯，k（cat）/K（M）为10（4）-10（5）M-1 s-1。 发现小鼠颗粒酶B具有天冬氨酸酶活性，并水解Boc-Ala-Ala-Asp-SBzl，k（cat）/K（M）值为2.3 × 10（5）M-1 s-1。当用CaCl 2代替测定中的0.05 M NaCl时，速率加快1.4倍。 制备的颗粒酶B对Boc-Ala-Ala-AA-SBzl底物也具有显著的活性，其中AA为Asn、Met或Ser [k（cat）/K（M）=（4-5）× 10（4）M-1 s-1]。 小鼠颗粒酶C，D和E没有水解任何硫酯底物，但含有对精氨酸或赖氨酸含硫酯的轻微污染活性。 鼠颗粒酶F对Suc-Phe-Leu-Phe-SBzl具有小的活性，沿着一些污染的胰蛋白酶样活性。 人Q31颗粒酶A，鼠，和人颗粒酶A被抑制相当有效的机制为基础的异香豆素抑制剂取代的碱性基团（胍基或异硫脲基丙氧基）。 虽然一般的丝氨酸蛋白酶抑制剂3，4-二氯异香豆素（DCI）对这些胰蛋白酶的灭活效果不佳，但它是对小鼠颗粒酶B的最佳异香豆素抑制剂（k（obs）/[I] = 3700-4200 M-1 s-1）。 鼠和人颗粒酶B也被Boc-Ala-Ala-Asp-CH 2Cl抑制;但抑制作用不如DCI。 DCI、3-（3-氨基-丙氧基）-4-氯异香豆素、4-氯-3-（3-异硫脲基丙氧基）异香豆素和7-氨基-4-氯-3-（3-异硫脲基丙氧基）异香豆素抑制Q31细胞毒性T淋巴细胞介导的人JY淋巴母细胞裂解（ED 50 = 0.5-5.0 μ M）。
The active site structures of human Q31 granzyme A, murine granzymes (A, B, C, D, E, and F), and human granzymes (A, B, and 3) isolated from cytotoxic T lymphocytes (CTL) were studied with peptide thioester substrates, peptide chloromethyl ketone, and isocoumarin inhibitors. Human Q31, murine, and human granzyme A hydrolyzed Arg- or Lys-containing thioesters very efficiently with k(cat)/K(M) of 10(4)-10(5) M-1 s-1. Murine granzyme B was found to have Asp-ase activity and hydrolyzed Boc-Ala-Ala-Asp-SBzl with a k(cat)/K(M) value of 2.3 X 10(5) M-1 s-1. The rate was accelerated 1.4-fold when the 0.05 M NaCl in the assay was replaced with CaCl2. The preparation of granzyme B also had significant activity toward Boc-Ala-Ala-AA-SBzl substrates, where AA was Asn, Met, or Ser [k(cat)/K(M) = (4-5) X 10(4) M-1 s-1]. Murine granzymes C, D, and E did not hydrolyze any thioester substrate but contained minor contaminating activity toward Arg- or Lys-containing thioesters. Murine granzyme F had small activity toward Suc-Phe-Leu-Phe-SBzl, along with some contaminating trypsin-like activity. Human Q31 granzyme A, murine, and human granzyme A were inhibited quite efficiently by mechanism-based isocoumarin inhibitors substituted with basic groups (guanidino or isothiureidopropoxy). Although the general serine protease inhibitor 3,4-dichloroisocoumarin (DCI) inactivated these tryptases poorly, it was the best isocoumarin inhibitor for murine granzyme B (k(obs)/[I] = 3700-4200 M-1 s-1). Murine and human granzyme B were also inhibited by Boc-Ala-Ala-Asp-CH2Cl; however, the inhibition was less potent than that with DCI. DCI, 3-(3-amino-propoxy)-4-chloroisocoumarin, 4-chloro-3-(3-isothiureidopropoxy)isocoumarin, and 7-amino-4-chloro-3-(3-isothiureidopropoxy)isocourmarin inhibited Q31 cytotoxic T lymphocyte mediated lysis of human JY lymphoblasts (ED50 = 0.5-5.0-mu-M).