Komissarov Andrey A, Declerck Paul J, Shore Joseph D
Division of Biochemical Research, Department of Pathology, Henry Ford Health System, Detroit, Michigan 48202, USA.
J Biol Chem. 2004 May 28;279(22):23007-13. doi: 10.1074/jbc.M401383200. Epub 2004 Mar 19.
Stopped-flow fluorometry was used to study the kinetics of the reactive center loop insertion occurring during the reaction of N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-3-diazole (NBD) P9 plasminogen activator inhibitor-1 (PAI-1) with tissue-(tPA) and urokinase (uPA)-type plasminogen activators and human pancreatic elastase at pH 5.5-8.5. The limiting rate constants of reactive center loop insertion (k(lim)) and concentrations of proteinase at half-saturation (K(0.5)) for tPA and uPA and the specificity constants (k(lim)/K(0.5)) for elastase were determined. The pH dependences of k(lim)/K(0.5) reflected inactivation of each enzyme due to protonation of His57 of the catalytic triad. However, the specificity of the inhibitory reaction with tPA and uPA was notably higher than that for the substrate reaction catalyzed by elastase. pH dependences of k(lim) and K(0.5) obtained for tPA revealed an additional ionizable group (pKa, 6.0-6.2) affecting the reaction. Protonation of this group resulted in a significant increase in both k(lim) and K(0.5) and a 4.6-fold decrease in the specificity of the reaction of tPA with NBD P9 PAI-1. Binding of monoclonal antibody MA-55F4C12 to PAI-1 induced a decrease in k(lim) and K(0.5) at any pH but did not affect either the pKa of the group or an observed decrease in k(lim)/K(0.5) due to protonation of the group. In contrast to tPA, the k(lim) and K(0.5) for the reactions of uPA with NBD P9 PAI-1 or its complex with the monoclonal antibody were independent of pH in the 6.5-8.5 range. Since slightly acidic pH is a feature of a number of malignant tumors, alterations in PAI-1/tPA kinetics could play a role in the cancerogenesis. Changes in the protonation state of His(188), which is placed closely to the S1 site and is unique for tPA, has been proposed to contribute to the observed pH dependences of k(lim) and K(0.5).
采用停流荧光法研究了N-((2-(碘乙酰氧基)乙基)-N-甲基)氨基-7-硝基苯并-2-恶唑-3-二氮杂萘（NBD）P9纤溶酶原激活物抑制剂-1（PAI-1）与组织型纤溶酶原激活物（tPA）、尿激酶型纤溶酶原激活物（uPA）以及人胰腺弹性蛋白酶在pH 5.5 - 8.5条件下反应时活性中心环插入的动力学过程。测定了tPA和uPA的活性中心环插入极限速率常数（k(lim)）以及半饱和时蛋白酶的浓度（K(0.5)），并测定了弹性蛋白酶的特异性常数（k(lim)/K(0.5)）。k(lim)/K(0.5)的pH依赖性反映了由于催化三联体中His57质子化导致每种酶失活。然而，与tPA和uPA的抑制反应特异性明显高于弹性蛋白酶催化的底物反应特异性。tPA的k(lim)和K(0.5)的pH依赖性揭示了一个影响反应的额外可电离基团（pKa，6.0 - 6.2）。该基团的质子化导致k(lim)和K(0.5)均显著增加，且tPA与NBD P9 PAI-1反应的特异性降低了4.6倍。单克隆抗体MA-55F4C12与PAI-1的结合在任何pH下均导致k(lim)和K(0.5)降低，但不影响该基团的pKa，也不影响因该基团质子化导致的k(lim)/K(0.5)降低。与tPA不同，uPA与NBD P9 PAI-1或其与单克隆抗体复合物反应的k(lim)和K(0.5)在6.5 - 8.5范围内与pH无关。由于许多恶性肿瘤具有微酸性pH特征，PAI-1/tPA动力学的改变可能在肿瘤发生中起作用。有人提出，紧邻S1位点且tPA特有的His(188)质子化状态的变化有助于观察到的k(lim)和K(0.5)的pH依赖性。
