Christensen S, Sottrup-Jensen L, Christensen U
Department of Molecular Biology, University of Aarhus, Denmark.
Biochem J. 1995 Jan 1;305 ( Pt 1)(Pt 1):97-102. doi: 10.1042/bj3050097.
In the conversion of bovine plasminogen to bovine plasmin not only the expected urokinase-catalysed cleavage of Arg-557-Val-558, and the following autocatalytic cleavage separating the N-terminal peptide 1-77 from the heavy chain of plasmin, but also a cleavage at Arg-342-Met-343 between kringles 3 and 4 is seen. Here, kinetic studies of the interaction of bovine alpha 2-antiplasmin with bovine plasmin were performed on isolated bovine midiplasmin (lacking kringles 1-3) and on bovine plasmin containing all of the activation products from the bovine plasminogen. A series of experiments using stopped-flow fluorescence fast kinetics as well as conventional techniques suggests a reaction model in accordance with the one known for the human system. First, a tight complex (K1 in the nanomolar range) is formed in a fast reaction step; and second, a tightening of this complex occurs in a slow reaction step. The final complex is indeed so tight (Ki < or = pM), that the reaction for many practical purposes is legitimately considered irreversible. The stopped-flow method allows for the determination of reliable values of the second-order rate constant for the fast association step. At pH 7.4 and 25 degrees C, k+1 = 1.7 x 10(6) M-1 s-1 was obtained in the absence and k+1 = 0.9 x 10(6) M-1.s-1 in the presence of the kringles 1-3 domain of bovine plasmin. In contrast to this, substantial reductions of k+1 were seen in the presence of concentrations of 6-amino-hexanoic acid corresponding to lysine-binding-site interactions and far too low to be attributed to active-site interactions with the bovine plasmins (for each, Ki = 42 mM). All in all, the data indicated that the lysine-binding site(s) not of kringle 1, but of midiplasmin (those of kringles 4 and 5) are regulating the inhibition reaction.
在将牛纤溶酶原转化为牛纤溶酶的过程中，不仅观察到了预期的尿激酶催化的精氨酸-557-缬氨酸-558裂解，以及随后将N端肽1-77与纤溶酶重链分离的自催化裂解，还观察到了在kringles 3和4之间精氨酸-342-甲硫氨酸-343处的裂解。在此，对分离的牛中纤溶酶（缺乏kringles 1-3）和含有来自牛纤溶酶原的所有激活产物的牛纤溶酶进行了牛α2-抗纤溶酶与牛纤溶酶相互作用的动力学研究。一系列使用停流荧光快速动力学以及传统技术的实验表明，反应模型与人类系统已知的模型一致。首先，在快速反应步骤中形成紧密复合物（K1在纳摩尔范围内）；其次，该复合物在缓慢反应步骤中发生紧密化。最终复合物确实非常紧密（Ki≤皮摩尔），以至于在许多实际应用中该反应可合理地视为不可逆。停流方法允许确定快速缔合步骤的二级速率常数的可靠值。在pH 7.4和25℃下，在不存在牛纤溶酶kringles 1-3结构域的情况下，k+1 = 1.7×10(6) M-1 s-1，在存在该结构域的情况下，k+1 = 0.9×10(6) M-1.s-1。与此相反，在存在对应于赖氨酸结合位点相互作用的6-氨基己酸浓度时，观察到k+1大幅降低，且该浓度远低于可归因于与牛纤溶酶活性位点相互作用的浓度（每种情况下，Ki = 42 mM）。总而言之，数据表明不是kringle 1的赖氨酸结合位点，而是中纤溶酶的（kringles 4和5的）赖氨酸结合位点在调节抑制反应。
