Matrix metalloproteinase-1 takes advantage of the induced fit mechanism to cleave the triple-helical type I collagen molecule.
Matrix metalloproteinase-1 takes advantage of the induced fit mechanism to cleave the triple-helical type I collagen molecule.
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DOI：
10.1021/bi060849d
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发表时间：
2006-12
期刊：
影响因子：
2.9
通讯作者：
T. O'Farrell;R. Guo;H. Hasegawa;T. Pourmotabbed
中科院分区：
文献类型：
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作者：
T. O'Farrell;R. Guo;H. Hasegawa;T. Pourmotabbed
胶原酶是基质金属蛋白酶家族 (MMP) 的成员，可降解天然三螺旋 I 型胶原蛋白。为了了解这些酶识别和裂解该底物的机制，我们研究了修饰形式的 MMP-1 (FC) 的底物特异性，其中其活性位点区域（氨基酸 212-254）已被 MMP-9 的活性位点区域（氨基酸 395-437）取代。尽管这种取代使酶对明胶和肽底物 Mca-PLGL(Dpa)AR-NH2 的活性分别增加了约 3 倍和约 11 倍，但它使酶的 I 型胶原分解活性降低至 0.13%。 Gly233（FC 该区域中所有胶原酶家族成员中唯一保守的氨基酸）被 MMP-9 中相应的 Glu 残基取代，导致该酶的 I 型胶原蛋白水解活性显着降低，但不影响其一般蛋白水解活性。 FC/G233E 突变体对于胶原底物的动力学参数与嵌合酶的动力学参数相似。此外，用 Gly233 代替嵌合体中的 Glu，酶的胶原蛋白分解活性增加了 12 倍。有趣的是，用 FC 中相应的残基 Gly 取代 MMP-9 中的 Glu415，赋予该酶具有 I 型胶原分解活性。 MMP-9突变体对三螺旋I型胶原的催化活性比胶原酶嵌合体高2倍。这些数据与 FC 的 X 射线晶体结构相结合表明，Gly233 提供了酶活性位点在底物结合时改变构象所需的灵活性。甘氨酸残基提供的灵活性对于 I 型胶原蛋白溶解活性至关重要。
The collagenases are members of the matrix metalloproteinase family (MMP) that degrade native triple-helical type I collagen. To understand the mechanism by which these enzymes recognize and cleave this substrate, we studied the substrate specificity of a modified form of MMP-1 (FC) in which its active site region (amino acids 212-254) had been replaced with that of MMP-9 (amino acids 395-437). Although this substitution increased the activity of the enzyme toward gelatin and the peptide substrate Mca-PLGL(Dpa)AR-NH2 by approximately 3- and approximately 11-fold, respectively, it decreased the type I collagenolytic activity of the enzyme to 0.13%. The replacement of Gly233, the only amino acid in this region of FC that is conserved in all collagenase family members, with the corresponding Glu residue in MMP-9 resulted in a substantial decrease in the type I collagenolytic activity of the enzyme without affecting its general proteolytic activities. The kinetic parameters of the FC/G233E mutant for the collagen substrate were similar to those of the chimeric enzyme. In addition, substituting Gly233 for Glu in the chimera increased the collagenolytic activity of the enzyme by 12-fold. Interestingly, replacing Glu415 in MMP-9 with Gly, its corresponding residue in FC, endowed the enzyme with type I collagenolytic activity. The catalytic activity of the MMP-9 mutant toward triple-helical type I collagen was 2-fold higher than that of the collagenase chimera. These data in conjunction with the X-ray crystal structure of FC indicate that Gly233 provides the flexibility necessary for the enzyme active site to change conformation upon substrate binding. The flexibility provided by the Gly residue is essential for type I collagenolytic activity.