Blouse Grant E, Perron Michel J, Kvassman Jan-Olov, Yunus Saadia, Thompson Jannah H, Betts Russell L, Lutter Leonard C, Shore Joseph D
Department of Pathology, Division of Biochemical Research, Henry Ford Health Sciences Center, Detroit, Michigan 48202-2689, USA.
Biochemistry. 2003 Oct 28;42(42):12260-72. doi: 10.1021/bi034737n.
We have demonstrated that interactions within the conserved serpin breach region play a direct role in the critical step of the serpin reaction in which the acyl-enzyme intermediate must first be exposed to hydrolyzing water and aqueous deacylation. Substitution of the breach tryptophan in PAI-1 (Trp175), a residue found in virtually all known serpins, with phenylalanine altered the kinetics of the reaction mechanism and impeded the ability of PAI-1 to spontaneously become latent without compromising the inherent rate of cleaved loop insertion or partitioning between the final inhibited serpin-proteinase complex and hydrolyzed serpin. Kinetic dissection of the PAI-1 inhibitory mechanism using multiple target proteinases made possible the identification of a single rate-limiting intermediate step coupled to the molecular interactions within the breach region. This step involves the initial insertion of the proximal reactive center loop hinge residue(s) into beta-sheet A and facilitates translocation of the distal P'-side of the cleaved reactive center loop from the substrate cleft of the proteinase. Substitution of the tryptophan residue raised the kinetic barrier restricting the initial loop insertion event, significantly retarding the rate-limiting step in tPA reactions in which strong exosite interactions must be overcome for the reaction to proceed.
我们已经证明，在保守的丝氨酸蛋白酶抑制剂（serpin）裂隙区域内的相互作用，在丝氨酸蛋白酶抑制剂反应的关键步骤中发挥直接作用，在该步骤中，酰基酶中间体必须首先暴露于水解水和水性脱酰基反应中。将几乎所有已知丝氨酸蛋白酶抑制剂中都存在的PAI-1（Trp175）中的裂隙色氨酸替换为苯丙氨酸，改变了反应机制的动力学，并阻碍了PAI-1自发潜伏的能力，同时不影响裂解环插入的固有速率或最终抑制性丝氨酸蛋白酶抑制剂 - 蛋白酶复合物与水解丝氨酸蛋白酶抑制剂之间的分配。使用多种靶蛋白酶对PAI-1抑制机制进行动力学剖析，使得能够识别与裂隙区域内分子相互作用相关的单个限速中间步骤。该步骤涉及近端反应中心环铰链残基最初插入β-折叠A中，并促进裂解的反应中心环的远端P'侧从蛋白酶的底物裂隙中移位。色氨酸残基的替换提高了限制初始环插入事件的动力学障碍，显著延迟了tPA反应中的限速步骤，在该反应中必须克服强烈的外部位点相互作用才能使反应进行。
