Schechter N M, Plotnick M, Selwood T, Walter M, Rubin H
Department of Dermatology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
J Biol Chem. 1997 Sep 26;272(39):24499-507. doi: 10.1074/jbc.272.39.24499.
Inhibition of human chymase by the serpins alpha1-antichymotrypsin (ACT) and alpha1-proteinase inhibitor (PI) at pH 8.0 produces a complex stable to dissociation by SDS/dithiothreitol and a second product, hydrolyzed/inactivated serpin. The first product is the presumed trapped acyl-enzyme complex typical of serpin inhibition, and the second is the result of a concurrent substrate-like reaction. As a result of the hydrolytic reaction, stoichiometries of inhibition (SI) appear greater than 1; values of 4 and 6.0 are observed for the chymase-ACT and -PI reactions. In this study the effect of pH on the inhibition rate constant (kinh) and the SI of each reaction were evaluated to better define the rate-limiting steps of the inhibitory and hydrolytic reaction pathways associated with chymase inhibition. Reactions were evaluated over a pH range to correlate kinh and SI with the ionizations (pK values of 7 and 9) that typically regulate serine protease catalytic activity. The results show that the effects of pH on SI and kinh differ for each inhibitor. On reducing the pH from 8.0 to 5.5, the chymase-ACT reaction exhibited a decrease in SI (to about 1) and little change in kinh, whereas the chymase-PI reaction revealed an increase in SI and a marked decrease in kinh. On increasing the pH from 8.0 to 10.0, the chymase-ACT reaction exhibited little change in SI and a marked decrease in kinh, whereas the chymase-PI reaction revealed a decrease in SI and a marked increase in kinh. Chymase catalytic properties determined for a peptide substrate were atypical over the high pH range exhibiting increases for kcat/Km and kcat and decreases for Km. This behavior suggests the presence of a high pH enzyme form with enhanced hydrolytic activity. From these results and others involving analyses of ACT/PI reactive loop chimeras and ACT point variants exhibiting a range of SI values, we suggest that the diverse pH effects on kinh and SI are caused largely by a difference in the abilities of ACT and PI to interact with low (catalytically inactive) and high (catalytically enhanced) pH forms of chymase. The constancy of kinh for the chymase-ACT reaction over the low pH range suggests that the rate-limiting step for inhibition is pH insensitive and not reflective of diminished chymase hydrolytic activity. Low pH did not appear to affect the rate of SDS-stable complex formation as complex accumulation, assessed qualitatively by SDS-PAGE, correlated with the loss of chymase enzymatic activity.
在pH 8.0条件下，丝氨酸蛋白酶抑制剂α1 - 抗糜蛋白酶（ACT）和α1 - 蛋白酶抑制剂（PI）对人糜蛋白酶的抑制作用会产生一种对SDS/二硫苏糖醇解离稳定的复合物以及第二种产物，即水解/失活的丝氨酸蛋白酶抑制剂。第一种产物是丝氨酸蛋白酶抑制剂抑制作用中典型的假定捕获酰基 - 酶复合物，第二种产物是同时发生的类似底物反应的结果。由于水解反应，抑制化学计量比（SI）似乎大于1；在糜蛋白酶 - ACT和 - PI反应中观察到的值分别为4和6.0。在本研究中，评估了pH对每个反应的抑制速率常数（kinh）和SI的影响，以更好地确定与糜蛋白酶抑制相关的抑制和水解反应途径的限速步骤。在一定pH范围内评估反应，以将kinh和SI与通常调节丝氨酸蛋白酶催化活性的电离作用（pK值为7和9）相关联。结果表明，每种抑制剂的pH对SI和kinh的影响不同。将pH从8.0降至5.5时，糜蛋白酶 - ACT反应的SI降低（至约1）且kinh变化不大，而糜蛋白酶 - PI反应的SI增加且kinh显著降低。将pH从8.0升至10.0时，糜蛋白酶 - ACT反应的SI变化不大且kinh显著降低，而糜蛋白酶 - PI反应的SI降低且kinh显著增加。针对肽底物测定的糜蛋白酶催化特性在高pH范围内是非典型的，表现为kcat/Km和kcat增加而Km降低。这种行为表明存在具有增强水解活性的高pH酶形式。根据这些结果以及其他涉及对ACT/PI反应性环嵌合体和表现出一系列SI值的ACT点变体的分析结果，我们认为pH对kinh和SI的不同影响主要是由于ACT和PI与糜蛋白酶的低（催化无活性）和高（催化增强）pH形式相互作用的能力差异所致。在低pH范围内，糜蛋白酶 - ACT反应的kinh恒定，这表明抑制的限速步骤对pH不敏感，且不反映糜蛋白酶水解活性的降低。低pH似乎不影响SDS稳定复合物的形成速率，因为通过SDS - PAGE定性评估的复合物积累与糜蛋白酶酶活性的丧失相关。
