The roles of substrate thermal stability and P2 and P1′ subsite identity on matrix metalloproteinase triple-helical peptidase activity and collagen specificity
The roles of substrate thermal stability and P2 and P1′ subsite identity on matrix metalloproteinase triple-helical peptidase activity and collagen specificity
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DOI：
10.1074/jbc.m606004200
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发表时间：
2006-12-15
影响因子：
4.8
通讯作者：
Fields, Gregg B.
中科院分区：
文献类型：
--
作者：
Minond, Dmitriy;Lauer-Fields, Janelle L.;Fields, Gregg B.
胶原蛋白的水解（胶原溶解）是细胞外基质周转的关键步骤之一。在基质金属蛋白酶（MMP）家族中，可以看到对胶原类型的不同偏好。可能指导这些特异性的底物决定因素尚不清楚。在这项研究中，我们已经利用了12个三螺旋底物与10个基质金属蛋白酶的组合，以更好地定义对三重解旋酶活性和胶原特异性的底物序列和热稳定性的贡献。通常，发现MMP-13与MMP-8和MT 1-MMP（Delta 279-523）不同，因为增强的底物热稳定性对活性仅具有适度的影响，而与序列无关。与MMP-8和MT 1-MMP相比，该结果与MMP-13的独特胶原特异性相关，因为MMP-13有效地水解II型胶原，而MMP-8和MT 1-MMP在它们对I型胶原的偏好方面相似。反过来，MMP-1是在处理越来越热稳定的三螺旋的胶原蛋白溶解MMP的效率最低，因此有利于III型胶原蛋白，其具有相对灵活的切割位点。明胶酶（MMP-2和MMP-9（Delta 444-707））似乎不能加工更稳定的螺旋，因此在机制上不同于胶原溶解性MMP。MMPs的胶原特异性似乎是基于底物序列和热稳定性的组合。MMP突变体对三螺旋肽的水解分析表明，Tyr（210）在三螺旋结合和水解中起作用，但在增加热稳定性的加工三螺旋中不起作用。MMP活性位点和exosites的进一步探索，结合底物构象，可能被证明是有价值的额外的解剖胶原溶解和产量的信息，在设计更具选择性的MMP抑制剂。
The hydrolysis of collagen (collagenolysis) is one of the committed steps in extracellular matrix turnover. Within the matrix metalloproteinase (MMP) family distinct preferences for collagen types are seen. The substrate determinants that may guide these specificities are unknown. In this study, we have utilized 12 triple-helical substrates in combination with 10 MMPs to better define the contributions of substrate sequence and thermal stability toward triple helicase activity and collagen specificity. In general, MMP-13 was found to be distinct from MMP-8 and MT1-MMP(Delta 279-523), in that enhanced substrate thermal stability has only a modest effect on activity, regardless of sequence. This result correlates to the unique collagen specificity of MMP-13 compared with MMP-8 and MT1-MMP, in that MMP-13 hydrolyzes type II collagen efficiently, whereas MMP-8 and MT1-MMP are similar in their preference for type I collagen. In turn, MMP-1 was the least efficient of the collagenolytic MMPs at processing increasingly thermal stable triple helices and thus favors type III collagen, which has a relatively flexible cleavage site. Gelatinases (MMP-2 and MMP-9(Delta 444-707)) appear incapable of processing more stable helices and are thus mechanistically distinct from collagenolytic MMPs. The collagen specificity of MMPs appears to be based on a combination of substrate sequence and thermal stability. Analysis of the hydrolysis of triple-helical peptides by an MMP mutant indicated that Tyr(210) functions in triple helix binding and hydrolysis, but not in processing triple helices of increasing thermal stabilities. Further exploration of MMP active sites and exosites, in combination with substrate conformation, may prove valuable for additional dissection of collagenolysis and yield information useful in the design of more selective MMP inhibitors.