Windsor L J, Steele D L, LeBlanc S B, Taylor K B
Research Center in Oral Biology, University of Alabama at Birmingham, 35294, USA.
Biochim Biophys Acta. 1997 Mar 15;1334(2-3):261-72. doi: 10.1016/s0304-4165(96)00102-x.
The propeptide plus the catalytic domain of human fibroblast-type collagenase, stromelysin-1, and matrilysin were expressed in Escherichia coli to directly compare the properties of all three catalytic domains utilizing the same assays. Truncated fibroblast-type collagenase (mini-CL), truncated stromelysin-1 (mini-SL-1), and matrilysin, like their native counterparts, could be activated by organomercurials, trypsin, or SDS. The mini-CL and mini-SL-1 displayed catalytic properties similar to their native counterparts, except that the mini-CL could not cleave native type I collagen. The k(cat)/Km for matrilysin (355 microM(-1) h(-1)) on the synthetic Mca-peptide was much higher than that for mini-CL (69 microM(-1) h(-1)) or mini-SL-1 (23.6 microM(-1) h(-1)). Mini-SL-1 and matrilysin, but not mini-CL, were capable of superactivating collagenase thus increasing the rate of collagen cleavage. Mini-CL and mini-SL-1, but not matrilysin, were able to form SDS-stable complexes with TIMP-1 when co-incubated with an organomercurial and TIMP-1. The second-order rate constant (k(on)) for TIMP-1 inhibition of mini-CL and mini-SL-1 were similar, 0.635 x 10(5) M(-1) s(-1) and 1.52 x 10(5) M(-1) s(-1), respectively. The k(on) for TIMP-1 inhibition of matrilysin was lower (0.130 x 10(5) M(-1) s(-1)) supporting the observation that no SDS stable complexes were detected. This study demonstrates that these catalytic domains are distinct and play a major role in the specificity of these enzymes in regard to rate of catalysis, TIMP-1 binding, and superactivation of collagenase.
将人成纤维细胞型胶原酶、基质溶解素-1和基质溶素的前肽加上催化结构域在大肠杆菌中表达，以便利用相同的检测方法直接比较这三种催化结构域的特性。截短的成纤维细胞型胶原酶（微型胶原酶，mini-CL）、截短的基质溶解素-1（微型基质溶解素-1，mini-SL-1）和基质溶素，与其天然对应物一样，可被有机汞化合物、胰蛋白酶或十二烷基硫酸钠（SDS）激活。微型胶原酶和微型基质溶解素-1表现出与其天然对应物相似的催化特性，只是微型胶原酶不能切割天然I型胶原。基质溶素对合成的Mca-肽的催化常数与米氏常数之比（k(cat)/Km，355 μM⁻¹ h⁻¹）远高于微型胶原酶（69 μM⁻¹ h⁻¹）或微型基质溶解素-1（23.6 μM⁻¹ h⁻¹）。微型基质溶解素-1和基质溶素，但不是微型胶原酶，能够超激活胶原酶，从而提高胶原的切割速率。微型胶原酶和微型基质溶解素-1，但不是基质溶素，在与有机汞化合物和金属蛋白酶组织抑制剂-1（TIMP-1）共同孵育时，能够与TIMP-1形成SDS稳定复合物。TIMP-1抑制微型胶原酶和微型基质溶解素-1的二级速率常数（k(on)）相似，分别为0.635×10⁵ M⁻¹ s⁻¹和1.52×10⁵ M⁻¹ s⁻¹。TIMP-1抑制基质溶素的k(on)较低（0.130×10⁵ M⁻¹ s⁻¹），这支持了未检测到SDS稳定复合物的观察结果。这项研究表明，这些催化结构域是不同的，并且在这些酶关于催化速率、TIMP-1结合和胶原酶超激活的特异性方面起主要作用。
