Knäuper V, Smith B, López-Otin C, Murphy G
Department of Cell and Molecular Biology, Strangeways Research Laboratory, Cambridge, UK.
Eur J Biochem. 1997 Sep 1;248(2):369-73. doi: 10.1111/j.1432-1033.1997.00369.x.
Human progelatinase B was activated by collagenase-3 in a time-dependent fashion. Activation proceeded through an intermediate form of Mr 86,000 to the final active form of Mr 82,000. N-terminal amino acid sequence determination demonstrated that the Glu40-Met41 peptide bond was initially hydrolysed followed by cleavage of the Arg87-Phe88 peptide bond releasing the rest of the propeptide domain which was accompanied by the achievement of maximal enzymatic activity as revealed using a quenched fluorescent substrate. Kinetic analysis of activation revealed that the rates were dependent on the concentration of the proenzyme as well as active collagenase-3. Active gelatinase B did not contribute to the activation rate of the proenzyme initiated by collagenase-3 and our results indicate that progelatinase B activation proceeds via bimolecular cleavage with collagenase-3 involving sequential cleavage of the propeptide in two steps. The activation rates were not dependent on C-terminal domain interactions between progelatinase B and collagenase-3, as assessed using wild-type and C-terminal deletion mutants of both enzymes. Since elevated levels of both gelatinase B and collagenase-3 have been observed in arthritis and breast cancer pathology these enzymes may well form a proteolytic cascade in these diseases which allows rapid turnover of the extracellular matrix.
人源前明胶酶B可被胶原酶-3以时间依赖性方式激活。激活过程通过86,000道尔顿的中间形式进行，最终形成82,000道尔顿的活性形式。N端氨基酸序列测定表明，最初水解的是Glu40-Met41肽键，随后是Arg87-Phe88肽键的裂解，释放出前肽结构域的其余部分，同时使用淬灭荧光底物显示达到最大酶活性。激活的动力学分析表明，速率取决于酶原以及活性胶原酶-3的浓度。活性明胶酶B对胶原酶-3引发的酶原激活速率没有贡献，我们的结果表明，前明胶酶B的激活通过与胶原酶-3的双分子裂解进行，涉及前肽的两步顺序裂解。使用两种酶的野生型和C端缺失突变体评估，激活速率不依赖于前明胶酶B和胶原酶-3之间的C端结构域相互作用。由于在关节炎和乳腺癌病理中均观察到明胶酶B和胶原酶-3水平升高，这些酶很可能在这些疾病中形成蛋白水解级联反应，从而使细胞外基质快速周转。
