Hedstrom L, Moorman A R, Dobbs J, Abeles R H
Biochemistry. 1984 Apr 10;23(8):1753-9. doi: 10.1021/bi00303a026.
The benzoxazinones 2-ethoxy-4H-3,1- benzoxazin -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 X 10(5) M-1 min-1 and greater than 4 X 10(6) M-1 min-1, respectively. The inactivated enzyme recovers catalytic activity slowly, k = 2.3 X 10(-3) min-1 and 3.7 X 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 ortho-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 para-substituted analogues [ Caplow , M., & Jencks , W. P. (1962) Biochemistry 1, 883-893]. The inactivation of chymotrypsin by 2-methyl-4H-3,1- benzoxazin -4-one (1b) is an equilibrium process (kinact = 1 X 10(4) M-1 min-1 and Keq = 2 X 10(6) 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.(ABSTRACT TRUNCATED AT 250 WORDS)
苯并恶嗪酮2 - 乙氧基 - 4H - 3,1 - 苯并恶嗪 - 4 - 酮（1a）和2 - （三氟甲基） - 4H - 3,1 - 苯并恶嗪 - 4 - 酮（1d）可使胰凝乳蛋白酶失活。这种失活是化学计量的，速率常数分别为7×10⁵ M⁻¹ min⁻¹和大于4×10⁶ M⁻¹ min⁻¹。失活的酶缓慢恢复催化活性，k = 2.3×10⁻³ min⁻¹和3.7×10⁻² min⁻¹（pH 7.1）。当酶恢复催化活性时，用1a失活的酶会释放出2 - [N - （乙氧羰基）氨基]苯甲酸，用1d失活的酶会释放出N - （三氟乙酰）邻氨基苯甲酸。失活机制涉及活性位点丝氨酸对失活剂C - 4羰基的攻击，这导致环打开并形成邻位取代的苯甲酰胰凝乳蛋白酶，由于取代基的电子释放能力，其水解缓慢。由1a或1d生成的苯甲酰胰凝乳蛋白酶的水解速率与根据它们对位取代类似物水解的哈米特参数（σ，ρ）预测的速率密切一致[卡普洛，M.，& 詹克斯，W. P.（1962年）《生物化学》1，883 - 893]。2 - 甲基 - 4H - 3,1 - 苯并恶嗪 - 4 - 酮（1b）使胰凝乳蛋白酶失活是一个平衡过程（失活常数 = 1×10⁴ M⁻¹ min⁻¹，平衡常数 = 2×10⁶ M⁻¹）。通过光谱变化和甲醇捕获证明了苯甲酰胰凝乳蛋白酶的形成。苯甲酰胰凝乳蛋白酶也可通过直接水解降解为N - 乙酰邻氨基苯甲酸。（摘要截断于250字）
