Wohl R C, Sinio L, Summaria L, Robbins K C
Biochim Biophys Acta. 1983 May 30;745(1):20-31. doi: 10.1016/0167-4838(83)90165-6.
Five native mammalian plasminogen species, namely, cat, dog, bovine, rabbit and horse, were studied and compared to native human plasminogen with respect to their substrate and enzymatic properties in various activated forms. These studies are an extension of previous work and were designed to confirm our previously proposed mechanism of plasminogen activation, using a series of native, but different, plasminogen substrates. The plasminogen activator species used were high molecular weight urokinase, streptokinase, human Glu-plasminogen-streptokinase complex, human plasmin-derived light(B)-chain-streptokinase complex, and the equimolar streptokinase activator complexes prepared from cat and dog plasmins. The peptidase parameters of the plasmins, plasmin-streptokinase and plasminogen-streptokinase complexes were determined with H-D-valyl-L-leucyl-L-lysyl-p-nitroanilide and Tos-glycyl-L-prolyl-L-lysyl-p-nitroanilide. Activation kinetics were measured with the same substrates. The peptidase parameters of all plasmin species were found to be similar, but with minor variations. The equimolar streptokinase mixtures of bovine, rabbit and horse plasminogens and plasmins did not form complexes and did not form active sites with plasminogen, under the conditions used. The second-order rate constants of activation revealed great differences (as much as 1400-fold), presumably expressing differences in the tertiary structure of the various plasminogen scissile bonds. The catalytic rate constants of activation, kplg, varied by as much as a 100-fold, while differences in Kplg were relatively small. The results of this study confirm the activation mechanism we have postulated previously, namely, that rapid-equilibrium rather than steady-state conditions prevail and that k2 (acylation) is the catalytic rate constant and the rate-determining step, while KS is a true dissociation constant. Calculations of the free energy of interaction of the peptidase and plasminogen activation reactions showed -4.4 to -5.6 kcal/mol for peptidase and -6.5 to -10 kcal/mol for the activation reaction. These values indicate 1-3 subsite binding interactions for the peptidase activity and 3-5 subsite binding interactions for the activation catalytic event. Streptokinase activator complexes have at least one more interacting subsite than the urokinase active site.
对五种天然哺乳动物纤溶酶原，即猫、狗、牛、兔和马的纤溶酶原进行了研究，并将其与天然人纤溶酶原在各种活化形式下的底物和酶学性质进行了比较。这些研究是先前工作的扩展，旨在利用一系列天然但不同的纤溶酶原底物来证实我们先前提出的纤溶酶原激活机制。所使用的纤溶酶原激活剂种类有高分子量尿激酶、链激酶、人谷氨酸纤溶酶原 - 链激酶复合物、人纤溶酶衍生的轻链（B） - 链激酶复合物，以及由猫和狗的纤溶酶制备的等摩尔链激酶激活剂复合物。用H - D - 缬氨酰 - L - 亮氨酰 - L - 赖氨酰 - 对硝基苯胺和甲苯磺酰 - 甘氨酰 - L - 脯氨酰 - L - 赖氨酰 - 对硝基苯胺测定了纤溶酶、纤溶酶 - 链激酶和纤溶酶原 - 链激酶复合物的肽酶参数。用相同的底物测量了激活动力学。发现所有纤溶酶种类的肽酶参数相似，但有微小差异。在所使用的条件下，牛、兔和马的纤溶酶原和纤溶酶的等摩尔链激酶混合物不形成复合物，也不与纤溶酶原形成活性位点。激活的二级速率常数显示出很大差异（高达1400倍），推测这表达了各种纤溶酶原可裂解键三级结构的差异。激活的催化速率常数kplg变化高达100倍，而Kplg的差异相对较小。本研究结果证实了我们先前假设的激活机制，即快速平衡而非稳态条件占主导，k2（酰化）是催化速率常数和速率决定步骤，而KS是真正的解离常数。肽酶和纤溶酶原激活反应相互作用自由能的计算表明，肽酶的自由能为 - 4.4至 - 5.6千卡/摩尔，激活反应的自由能为 - 6.5至 - 10千卡/摩尔。这些值表明肽酶活性有1 - 3个亚位点结合相互作用，激活催化事件有3 - 5个亚位点结合相互作用。链激酶激活剂复合物比尿激酶活性位点至少多一个相互作用亚位点。
