Urano T, Sator de Serrano V, Chibber B A, Castellino F J
Department of Chemistry, University of Notre Dame, Indiana 46556.
J Biol Chem. 1987 Nov 25;262(33):15959-64.
The urokinase-catalyzed activation of human Glu1-plasminogen (Glu1Pg) has been found to be inhibited by monovalent anions in the following order of effectiveness: I- greater than SCN- greater than Cl- greater than IO3- greater than HCOO- greater than F- greater than OAc-. The inhibition is reversed by epsilon-aminocaproic acid, with its effectiveness in this capacity generally inversely proportional to the strength of the binding of the anion. The physical basis for the anion inhibition and epsilon-aminocaproic acid stimulation lies in the ability of these effectors to cause measurable opposite alterations in the conformation of Glu1Pg, which are revealed through study of the sedimentation velocity of the protein under various conditions. The kinetic mechanism of the chloride inhibition of Glu1Pg activation has been examined in detail. It has been found that the Glu1Pg.Cl complex serves as an alternate substrate to Glu1Pg for urokinase, with a greatly increased Km (25 +/- 3 and 2.2 +/- 0.3 microM, respectively) for activation. The kcat for the urokinase.Glu1Pg.Cl complex is approximately the same as that for urokinase.Glu1Pg (1.6 +/- 0.2 - 2.0 +/- 0.2/s). Similarly, the stimulation by epsilon-aminocaproic acid also results from effects on the Km of the activation, which is reduced to 1.8 +/- 0.2 microM for the Glu1Pg.Cl.epsilon-aminocaproic acid complex. The kcat for the urokinase.Glu1Pg.Cl.epsilon-aminocaproic acid of 2.4 +/- 0.3/s complex is not greatly different from that for urokinase.Glu1Pg.Cl. Nuclear magnetic resonance studies of the Glu1Pg-induced line broadening of the 35Cl- spectra in the presence and absence of epsilon-aminocaproic acid suggest that Cl- and epsilon-aminocaproic acid simultaneously bind to the protein and that each of these effectors displays its effects through separate binding sites.
已发现单价阴离子可抑制尿激酶催化的人谷氨酸-1-纤溶酶原（Glu1Pg）的激活，其抑制效果顺序如下：I⁻＞SCN⁻＞Cl⁻＞IO₃⁻＞HCOO⁻＞F⁻＞OAc⁻。ε-氨基己酸可逆转这种抑制作用，其在这方面的效果通常与阴离子结合强度成反比。阴离子抑制和ε-氨基己酸刺激的物理基础在于这些效应物能够使Glu1Pg的构象发生可测量的相反变化，这通过研究蛋白质在各种条件下的沉降速度得以揭示。已详细研究了氯离子对Glu1Pg激活的抑制动力学机制。已发现Glu1Pg·Cl复合物作为尿激酶作用于Glu1Pg的替代底物，其激活的米氏常数（Km）大幅增加（分别为25±3和2.2±0.3微摩尔）。尿激酶·Glu1Pg·Cl复合物的催化常数（kcat）与尿激酶·Glu1Pg的大致相同（1.6±0.2 - 2.0±0.2/秒）。同样，ε-氨基己酸的刺激作用也源于对激活的Km的影响，对于Glu1Pg·Cl·ε-氨基己酸复合物，其Km降至1.8±0.2微摩尔。尿激酶·Glu1Pg·Cl·ε-氨基己酸复合物的kcat为2.4±0.3/秒，与尿激酶·Glu1Pg·Cl的相差不大。在有和没有ε-氨基己酸存在的情况下，对Glu1Pg诱导的³⁵Cl⁻光谱线加宽的核磁共振研究表明，Cl⁻和ε-氨基己酸同时与蛋白质结合，并且这些效应物各自通过独立的结合位点发挥作用。
