SUICIDE INACTIVATION OF CHYMOTRYPSIN BY BENZOXAZINONES
SUICIDE INACTIVATION OF CHYMOTRYPSIN BY BENZOXAZINONES
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DOI：
10.1021/bi00303a026
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发表时间：
1984-01-01
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：
HEDSTROM, L;MOORMAN, AR;ABELES, RH
苯并恶嗪2-乙氧基- 4h -3,1-苯并恶嗪-4-one （1a）和2-（三氟甲基）- 4h -3,1-苯并恶嗪-4-one （1d）灭活凝乳胰蛋白酶。失活是化学计量的，其速率常数为7。105次和40次。分别是106 M-1 min-1。失活酶恢复催化活性较慢，k = 2.3倍。10-3分钟-1和3.7次。10-2 min-1 （pH 7.1）。当酶恢复催化活性时，用1a灭活的酶释放2-[N-（乙氧羰基）氨基]苯甲酸，用1d灭活的酶释放N-（三氟乙酰）苯甲酸。失活的机制包括失活剂的活性位点丝氨酸攻击C-4羰基，导致环打开并形成o取代的苯甲酰胰糜蛋白酶，由于取代基的电子释放能力，该酶水解缓慢。从1a或1d中得到的苯甲酰胰凝乳蛋白酶的水解速率与从其p取代类似物的哈米特参数（.sigma.,.rho.）中预测的水解速率非常接近。2-甲基- 4h -3,1-苯并恶嗪-4-one （1b）对胰凝乳酶的失活是一个平衡过程(kinact = 1。104 M-1 min-1和Keq = 2。106 m - 1)。苯甲酰胰凝乳酶的形成通过光谱变化和甲醇捕获来证明。苯甲酰胰凝乳蛋白酶也可以直接水解为n -乙酰氨基苯甲酸而衰变。凝乳胰蛋白酶也被2-氨基- 4h -3,1-苯并恶嗪-4-one灭活（1c）。失活速度快（k = 8.7倍）。k = 0.14 min-1)，但再活化也相对较快。再活化后，苯甲酰脲被释放。1c与胰凝乳酶反应生成（o-脲基苯甲酰）-胰凝乳酶，该酶不被水解，而是通过脲基NH2在苯甲酰酯羰基上的分子内攻击而分解。化合物1a也灭活胰蛋白酶和弹性酶。化合物1b是一种较差的胰蛋白酶灭活剂，不抑制弹性酶。胰蛋白酶和弹性酶都能催化1c转化为苯甲酰脲。
The benzoxazines 2-ethoxy-4H-3,1-benzoxazine-4-one (1a) and 2-(trifluoromethyl)-4H-3,1-benzoxazin-4-one (1d) inactivate chymotrypsin. The inactivation is stoichiometric and proceeds with rate constants of 7 .times. 105 and > 4 .times. 106 M-1 min-1, respectively. The inactivated enzyme recovers catalytic activity slowly, k = 2.3 .times. 10-3 min-1 and 3.7 .times. 10-2 min-1 (pH 7.1). When the enzyme regains catalytic activity, 2-[N-(ethoxycarbonyl)amino]benzoic acid is released from enzyme inactivated with 1a and N-(trifluoroacetyl)anthranilic acid from enzyme inactivated with 1d. The mechanism of inactivation involves attack of the active site serine on the C-4 carbonyl of the inactivator which leads to ring opening and formation of an o-substituted benzoylchymotrypsin, which hydrolyzes slowly due to electron releasing ability of the substituents. The rate of hydrolysis of the benzoylchymotrypsin from 1a or 1d is in close agreement with those predicted from the Hammett parameters (.sigma.,.rho.) for hydrolysis of their p-substituted analog. The inactivation of chymotrypsin by 2-methyl-4H-3,1-benzoxazin-4-one (1b) is an equilibrium process (kinact = 1 .times. 104 M-1 min-1 and Keq = 2 .times. 106 M-1). Formation of a benzoylchymotrypsin is demonstrated by spectral changes and methanol trapping. The benzoylchymotrypsin can also decay by direct hydrolysis to N-acetylanthranilic acid. Chymotrypsin is also inactivated by 2-amino-4H-3,1-benzoxazin-4-one (1c). The inactivation is rapid (k = 8.7 .times. 104 M-1 min-1), but reactiativation is also relatively fast (k = 0.14 min-1). Upon reactivation benzoyleneurea is released. Reaction of 1c with chymotrypsin leads to the formation of (o-ureidobenzoyl)-chymotrypsin, which does not undergo hydrolysis but decomposes through intramolecular attack of the ureido NH2 upon the carbonyl group of the benzoyl ester. Compound 1a also inactivates trypsin and elastase. Compound 1b was a poor inactivator of trypsin and does not inhibit elastase. Both trypsin and elastase catalyze the conversion of 1c to benzoyleneurea.