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首页> 外文期刊>Biochemistry >Kinetic mechanism of the inhibition of cathepsin G by alpha(1)-antichymotrypsin and alpha(1)-proteinase inhibitor
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Kinetic mechanism of the inhibition of cathepsin G by alpha(1)-antichymotrypsin and alpha(1)-proteinase inhibitor

机译：α（1）-抗胰凝乳蛋白酶和α（1）-蛋白酶抑制剂抑制组织蛋白酶G的动力学机制

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Uncontrolled proteolysis due to cathepsin G (cat G) may cause severe pathological disorders. Cat G is inhibited by alpha(1)-antichymotrypsin (ACT) and alpha(1)-proteinase inhibitor (alpha(1)PI), two members of the serpin superfamily of proteins. To see whether these two inhibitors play a physiological proteolysis-preventing function, we have made a detailed kinetic investigation of their reaction with cat G, The kinetics of inhibition of cat G in the presence of inhibitor and substrate evidenced a two-step inhibition mechanism: [GRAPHICS] The cat G/ACT interaction is described by K-i* = 6.2 x 10(-8) M and k(2) = 2.8 x 10(-2) s(-1), while the cat G/alpha(1)PI association is governed by K-i* = 8.1 x 10(-7) M and k(2) = 5.5 x 10(-2) s(-1). The reliability of these kinetic constants was checked using a number of experiments which all gave consistent results: (i) both EI* complexes were found to be enzymatically inactive, (ii) the K-i* values were determined directly using initial velocity experiments of cat G-catalyzed hydrolysis of substrate in the presence of inhibitor, (iii) the second-order rate constants k(2)/K-i* were measured using second-order inhibition experiments in the absence of substrate, and (iv) the ratio of the two second-order rate constants was determined by measuring the partition of cat G between the two fluorescently labeled serpins. Since the plasma concentrations of ACT and alpha(1)PI are much higher than their K-i* values, cat G released from neutrophils will be fully taken up as rapidly forming EI* complexes, that is, 70% with ACT and 30% with c alpha(1)PI. Both ACT and alpha(1)PI are thus physiological cat G inhibitors whose inhibitory potential does not depend on the formation of the stable inhibitory species EI characteristic of serpins. Such an in vivo inhibition mechanism might take place with other serpin/proteinase systems. [References: 32]
机译：由于组织蛋白酶G（cat G）引起的不受控制的蛋白水解可能会导致严重的病理疾病。 Cat G被alpha（1）-抗胰凝乳蛋白酶（ACT）和alpha（1）-蛋白酶抑制剂（alpha（1）PI）抑制，这是丝氨酸蛋白酶抑制蛋白家族的两个成员。为了了解这两种抑制剂是否具有生理上的蛋白水解预防功能，我们对它们与cat G的反应进行了详细的动力学研究。在抑制剂和底物存在下对cat G的抑制动力学证明了两步抑制机理： [图形] cat G / ACT相互作用描述为Ki * = 6.2 x 10（-8）M和k（2）= 2.8 x 10（-2）s（-1），而cat G / alpha（1 PI关联由Ki * = 8.1 x 10（-7）M和k（2）= 5.5 x 10（-2）s（-1）控制。使用大量提供了一致结果的实验​​检查了这些动力学常数的可靠性：（i）发现两种EI *复合物均无酶活性;（ii）使用Cat G的初始速度实验直接确定Ki *值-在抑制剂存在下催化水解底物，（iii）在不存在底物的情况下，使用二级抑制实验测量了二级速率常数k（2）/ Ki *，（iv）两者之比通过测量两个荧光标记的丝氨酸蛋白酶抑制剂之间的cat G分配来确定二级速率常数。由于ACT和alpha（1）PI的血浆浓度远远高于其Ki *值，因此从中性粒细胞释放的cat G将作为快速形成的EI *复合物被完全吸收，即ACT为70％，c为30％ alpha（1）PI。因此，ACT和alpha（1）PI都是生理性Cat G抑制剂，其抑制潜能不取决于丝氨酸蛋白酶抑制剂的稳定抑制物种EI的形成。这种体内抑制机制可能与其他丝氨酸蛋白酶抑制剂/蛋白酶系统一起发生。 [参考：32]

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