Arza B, Hoylaerts M F, Félez J, Collen D, Lijnen H R
Center for Molecular and Vascular Biology, University of Leuven, Belgium.
Eur J Biochem. 2000 Nov;267(21):6378-84. doi: 10.1046/j.1432-1327.2000.01732.x.
Matrix metalloproteinase-3 (MMP-3 or stromelysin-1) specifically binds to tissue-type plasminogen activator (t-PA), without however, hydrolyzing the protein. Binding affinity to proMMP-3 is similar to single chain t-PA, two chain t-PA and active site mutagenized t-PA (Ka of 6.3 x 106 to 8.0 x 106 M-1), but is reduced for t-PA lacking the finger and growth factor domains (Ka of 2.0 x 106 M-1). Activation of native Glu-plasminogen by t-PA in the presence of proMMP-3 obeys Michaelis-Menten kinetics; at saturating concentrations of proMMP-3, the catalytic efficiency of two chain t-PA is enhanced 20-fold (kcat/Km of 7.9 x 10-3 vs. 4.1 x 10-4 microM-1.s-1). This is mainly the result of an enhanced affinity of t-PA for its substrate (Km of 1.6 microM vs. 89 microM in the absence of proMMP-3), whereas the kcat is less affected (kcat of 1.3 x 10-2 vs. 3.6 x 10-2 s-1). Activation of Lys-plasminogen by two chain t-PA is stimulated about 13-fold at a saturating concentration of proMMP-3, whereas that of miniplasminogen is virtually unaffected (1.4-fold). Plasminogen activation by single chain t-PA is stimulated about ninefold by proMMP-3, whereas that by the mutant lacking finger and growth factor domains is stimulated only threefold. Biospecific interaction analysis revealed binding of Lys-plasminogen to proMMP-3 with 18-fold higher affinity (Ka of 22 x 106 M-1) and of miniplasminogen with fivefold lower affinity (Ka of 0.26 x 106 M-1) as compared to Glu-plasminogen (Ka of 1.2 x 106 M-1). Plasminogen and t-PA appear to bind to different sites on proMMP-3. These data are compatible with a model in which both plasminogen and t-PA bind to proMMP-3, resulting in a cyclic ternary complex in which t-PA has an enhanced affinity for plasminogen, which may be in a Lys-plasminogen-like conformation. Maximal binding and stimulation require the N-terminal finger and growth factor domains of t-PA and the N-terminal kringle domains of plasminogen.
基质金属蛋白酶-3（MMP-3或基质溶解素-1）能特异性结合组织型纤溶酶原激活剂（t-PA），但不会水解该蛋白。其与前MMP-3的结合亲和力与单链t-PA、双链t-PA及活性位点诱变的t-PA相似（解离常数Ka为6.3×10⁶至8.0×10⁶ M⁻¹），但对于缺乏指状结构域和生长因子结构域的t-PA，其结合亲和力降低（解离常数Ka为2.0×10⁶ M⁻¹）。在存在前MMP-3的情况下，t-PA对天然谷氨酸纤溶酶原的激活遵循米氏动力学；在前MMP-3饱和浓度下，双链t-PA的催化效率提高了20倍（催化常数与米氏常数之比kcat/Km为7.9×10⁻³对比4.1×10⁻⁴ μM⁻¹·s⁻¹）。这主要是由于t-PA对其底物的亲和力增强（米氏常数Km为1.6 μM，而在不存在前MMP-3时为89 μM），而催化常数受影响较小（催化常数kcat为1.3×10⁻²对比3.6×10⁻² s⁻¹）。在饱和浓度的前MMP-3存在下，双链t-PA对赖氨酸纤溶酶原的激活刺激约13倍，而对微型纤溶酶原的激活几乎没有影响（1.4倍）。单链t-PA对纤溶酶原的激活受前MMP-3刺激约9倍，而缺乏指状结构域和生长因子结构域的突变体对纤溶酶原激活的刺激仅为3倍。生物特异性相互作用分析显示，与谷氨酸纤溶酶原（解离常数Ka为1.2×10⁶ M⁻¹）相比，赖氨酸纤溶酶原与前MMP-3的结合亲和力高18倍（解离常数Ka为22×10⁶ M⁻¹），微型纤溶酶原与前MMP-3的结合亲和力低5倍（解离常数Ka为0.26×10⁶ M⁻¹）。纤溶酶原和t-PA似乎结合在前MMP-3的不同位点上。这些数据与一种模型相符，即纤溶酶原和t-PA都与前MMP-3结合，形成一个环状三元复合物，其中t-PA对纤溶酶原的亲和力增强，纤溶酶原可能处于赖氨酸纤溶酶原样构象。最大结合和刺激需要t-PA的N端指状结构域和生长因子结构域以及纤溶酶原的N端kringle结构域。
