Minond Dmitriy, Lauer-Fields Janelle L, Cudic Mare, Overall Christopher M, Pei Duanqing, Brew Keith, Moss Marcia L, Fields Gregg B
Department of Chemistry and Biochemistry, Florida Atlantic University, 777 Glades Road, Boca Raton, Florida 33431-0991, USA.
Biochemistry. 2007 Mar 27;46(12):3724-33. doi: 10.1021/bi062199j. Epub 2007 Mar 6.
The turnover of the collagen triple-helical structure (collagenolysis) is a tightly regulated process in normal physiology and has been ascribed to a small number of proteases. Several members of the matrix metalloproteinase (MMPs) family possess collagenolytic activity, and the mechanisms by which these enzymes process triple helices are beginning to be unraveled. The present study has utilized two triple-helical sequences to compare the cleavage-site specificities of 10 MMPs. One substrate featured a continuous Gly-Xxx-Yyy sequence (Pro-Leu-Gly approximately Met-Arg-Gly), while the other incorporated an interruption in the Gly-Xxx-Yyy repeat (Pro-Val-Asn approximately Phe-Arg-Gly). Both sequences were selectively cleaved by MMP-13 while in linear form, but neither proved to be selective within a triple helix. This suggests that the conformational presentation of substrate sequences to a MMP active site is critical for enzyme specificity, in that activities differ when sequences are presented from an unwound triple helix versus an independent single strand. Differences in specificity between secreted and membrane-type (MT) MMPs were also observed for both sequences, where MMP-2 and MT-MMPs showed an ability to hydrolyze a triple helix at an additional site (Gly-Gln bond). Interruption of the triple helix had different effects on secreted MMPs and MT-MMPs, because MT-MMPs could not hydrolyze the Asn-Phe bond but instead cleaved the triple helix closer to the C terminus at a Gly-Gln bond. It is possible that MT-MMPs have a requirement for Gly in the P1 subsite to be able to efficiently process a triple-helical molecule. Analysis of individual kinetic parameters and activation energies indicated different substrate preferences within secreted MMPs, because MMP-13 preferred the interrupted sequence, while MMP-8 showed little discrimination between non-interrupted and interrupted triple helices. On the basis of the present and prior studies, we can assign unique triple-helical peptidase behaviors to the collagenolytic MMPs. Such differences may be significant for understanding MMP mechanisms of action and aid in the development of selective MMP inhibitors.
胶原蛋白三螺旋结构的周转（胶原降解）在正常生理过程中是一个受到严格调控的过程，并且已归因于少数几种蛋白酶。基质金属蛋白酶（MMPs）家族的几个成员具有胶原降解活性，这些酶处理三螺旋的机制正开始被阐明。本研究利用两个三螺旋序列来比较10种MMPs的切割位点特异性。一种底物具有连续的Gly-Xxx-Yyy序列（Pro-Leu-Gly近似于Met-Arg-Gly），而另一种在Gly-Xxx-Yyy重复序列中存在中断（Pro-Val-Asn近似于Phe-Arg-Gly）。两种序列在呈线性形式时均被MMP-13选择性切割，但在三螺旋结构中均未表现出选择性。这表明底物序列向MMP活性位点的构象呈现对于酶的特异性至关重要，因为当序列从未缠绕的三螺旋与独立的单链呈现时活性会有所不同。对于这两种序列，还观察到分泌型和膜型（MT）MMPs之间的特异性差异，其中MMP-2和MT-MMPs显示出在另一个位点（Gly-Gln键）水解三螺旋的能力。三螺旋的中断对分泌型MMPs和MT-MMPs有不同影响，因为MT-MMPs不能水解Asn-Phe键，而是在靠近C末端的Gly-Gln键处切割三螺旋。MT-MMPs可能需要P1亚位点中有Gly才能有效处理三螺旋分子。对各个动力学参数和活化能的分析表明分泌型MMPs内存在不同的底物偏好，因为MMP-13更喜欢中断的序列，而MMP-8对未中断和中断的三螺旋几乎没有区分。基于目前和先前的研究，我们可以为胶原降解性MMPs赋予独特的三螺旋肽酶行为。这种差异对于理解MMP的作用机制可能具有重要意义，并有助于开发选择性MMP抑制剂。
