Department of Periodontics, University of Texas Health Science Center, San Antonio, Texas 78229, USA.
J Biol Chem. 2011 Oct 28;286(43):37535-42. doi: 10.1074/jbc.M111.273391. Epub 2011 Sep 6.
Members of the matrix metalloproteinase (MMP) family selectively cleave collagens in vivo. However, the substrate structural determinants that facilitate interaction with specific MMPs are not well defined. We hypothesized that type I-III collagen sequences located N- or C-terminal to the physiological cleavage site mediate substrate selectivity among MMP-1, MMP-2, MMP-8, MMP-13, and MMP-14/membrane-type 1 (MT1)-MMP. The enzyme kinetics for hydrolysis of three fluorogenic triple-helical peptides (fTHPs) was evaluated herein. The first fTHP contained consensus residues 769-783 from type I-III collagens, the second inserted α1(II) collagen residues 763-768 N-terminal to the consensus sequence, and the third inserted α1(II) collagen residues 784-792 C-terminal to the consensus sequence. Our analyses showed that insertion of the C-terminal residues significantly increased k(cat)/K(m) and k(cat) for MMP-1. MMP-13 showed the opposite behavior with a decreased k(cat)/K(m) and k(cat) and a greatly improved K(m) in response to the C-terminal residues. Insertion of the N-terminal residues enhanced k(cat)/K(m) and k(cat) for MMP-8 and MT1-MMP. For MMP-2, the C-terminal residues enhanced K(m) and dramatically decreased k(cat), resulting in a decrease in the overall activity. These changes in activities and kinetic parameters represented the collagen preferences of MMP-8, MMP-13, and MT1-MMP well. Thus, interactions with secondary binding sites (exosites) helped direct the specificity of these enzymes. However, MMP-1 collagen preferences were not recapitulated by the fTHP studies. The preference of MMP-1 for type III collagen appears to be primarily based on the flexibility of the hydrolysis site of type III collagen compared with types I and II. Further characterization of exosite determinants that govern interactions of MMPs with collagenous substrates should aid the development of pharmacotherapeutics that target individual MMPs.
基质金属蛋白酶（MMP）家族成员在体内选择性地切割胶原蛋白。然而，促进与特定 MMP 相互作用的底物结构决定因素尚未很好地定义。我们假设位于生理切割位点 N-或 C-末端的 I 型-III 型胶原蛋白序列在 MMP-1、MMP-2、MMP-8、MMP-13 和 MMP-14/膜型 1（MT1）-MMP 之间介导底物选择性。本文评估了三种荧光三螺旋肽（fTHP）水解的酶动力学。第一个 fTHP 包含 I 型-III 型胶原蛋白的共有残基 769-783，第二个插入的 α1(II)胶原蛋白残基 763-768 在共有序列的 N-末端，第三个插入的 α1(II)胶原蛋白残基 784-792 在共有序列的 C-末端。我们的分析表明，插入 C 末端残基显著增加了 MMP-1 的 k(cat)/K(m)和 k(cat)。MMP-13 的行为则相反，k(cat)/K(m)和 k(cat)降低，K(m)大大提高，对 C 末端残基有反应。插入 N 末端残基增强了 MMP-8 和 MT1-MMP 的 k(cat)/K(m)和 k(cat)。对于 MMP-2，C 末端残基增强了 K(m)，并显著降低了 k(cat)，导致整体活性降低。这些活性和动力学参数的变化很好地代表了 MMP-8、MMP-13 和 MT1-MMP 的胶原蛋白偏好。因此，与次级结合位点（外显子）的相互作用有助于指导这些酶的特异性。然而，fTHP 研究并没有再现 MMP-1 的胶原蛋白偏好。MMP-1 对 III 型胶原蛋白的偏好似乎主要基于 III 型胶原蛋白水解位点与 I 型和 II 型相比的灵活性。进一步表征决定 MMP 与胶原蛋白底物相互作用的外显子决定因素，应有助于开发针对特定 MMP 的药物治疗。
