Tanizawa K, Kanaoka Y, Wos J D, Lawson W B
Biol Chem Hoppe Seyler. 1985 Sep;366(9):871-8. doi: 10.1515/bchm3.1985.366.2.871.
The interactions of human thrombin and bovine trypsin with 4-amidinophenylpyruvate (p-APPA), 3-amidinophenylpyruvate (m-APPA) and benzamidine were studied by equilibrium binding and by stopped-flow kinetics with proflavin displacement. The excellent inhibitory properties of p-APPA with both enzymes are explained by a two-step transition-state mechanism in which the initial Michaelis complex E.I reacts rapidly to form a fairly stable, chemically bonded complex E-I, in which the keto group of p-APPA forms a hemiketal with the gamma-O-atom of Ser195 at the active site. The hemiketal complex of thrombin and p-APPA may be further stabilized by a hydrogen bond between a carboxylate oxygen of p-APPA and the N tau-atom (= N epsilon 2) of His57, as was previously shown for the trypsin-p-APPA complex by X-ray crystal structure analysis by J. Walter and W. Bode. m-APPA is apparently sterically incapable of forming a hemiketal with Ser195 O gamma; it does not bind to thrombin or trypsin in a time-dependent manner, and it displays KI values with both enzymes close to those obtained for benzamidine itself. In the p-APPA-thrombin reaction the overall binding constant KI (E-I) is 1.3 microM, while the initial binding displays a KM (E.I) estimated at least 100-fold higher (700 microM). The half-time for the formation of E-I is about 0.6 s at a p-APPA concentration of 1 microM.
通过平衡结合以及采用原黄素置换的停流动力学方法，研究了人凝血酶和牛胰蛋白酶与4-脒基苯丙酮酸（p-APPA）、3-脒基苯丙酮酸（m-APPA）和苯甲脒的相互作用。p-APPA对这两种酶均具有出色的抑制特性，这可通过两步过渡态机制来解释，即初始的米氏复合物E·I迅速反应形成一种相当稳定的化学键合复合物E-I，其中p-APPA的酮基与活性位点处Ser195的γ-O原子形成半缩酮。凝血酶与p-APPA的半缩酮复合物可能通过p-APPA的羧酸盐氧与His57的Nτ原子（= Nε2）之间的氢键进一步稳定，正如J. Walter和W. Bode先前通过X射线晶体结构分析对胰蛋白酶-p-APPA复合物所显示的那样。m-APPA显然在空间上无法与Ser195 Oγ形成半缩酮；它不会以时间依赖性方式与凝血酶或胰蛋白酶结合，并且它与这两种酶的KI值接近苯甲脒自身的KI值。在p-APPA - 凝血酶反应中，总体结合常数KI（E-I）为1.3 μM，而初始结合的KM（E-I）估计至少高100倍（700 μM）。在p-APPA浓度为1 μM时，E-I形成的半衰期约为0.6秒。
