Proteolysis Laboratory, Department of Structural Biology, Molecular Biology Institute of Barcelona, Consejo Superior de Investigaciones Científicas, Barcelona Science Park, Helix Building, c/Baldiri Reixac 15-21, E-08028, Spain.
J Biol Chem. 2010 Apr 30;285(18):13958-65. doi: 10.1074/jbc.M109.097436. Epub 2010 Mar 4.
Proteolysis is regulated by inactive (latent) zymogens, with a prosegment preventing access of substrates to the active-site cleft of the enzyme. How latency is maintained often depends on the catalytic mechanism of the protease. For example, in several families of the metzincin metallopeptidases, a "cysteine switch" mechanism involves a conserved prosegment motif with a cysteine residue that coordinates the catalytic zinc ion. Another family of metzincins, the astacins, do not possess a cysteine switch, so latency is maintained by other means. We have solved the high resolution crystal structure of proastacin from the European crayfish, Astacus astacus. Its prosegment is the shortest structurally reported for a metallopeptidase, and it has a unique structure. It runs through the active-site cleft in reverse orientation to a genuine substrate. Moreover, a conserved aspartate, projected by a wide loop of the prosegment, coordinates the zinc ion instead of the catalytic solvent molecule found in the mature enzyme. Activation occurs through two-step limited proteolysis and entails major rearrangement of a flexible activation domain, which becomes rigid and creates the base of the substrate-binding cleft. Maturation also requires the newly formed N terminus to be precisely trimmed so that it can participate in a buried solvent-mediated hydrogen-bonding network, which includes an invariant active-site residue. We describe a novel mechanism for latency and activation, which shares some common features both with other metallopeptidases and with serine peptidases.
蛋白水解作用受无活性（潜伏）酶原的调节，其前导肽段阻止底物进入酶的活性部位裂隙。潜伏状态的维持通常取决于蛋白酶的催化机制。例如，在几种金属蛋白酶的 Metzincin 家族中，“半胱氨酸开关”机制涉及到一个保守的前导肽段结构，其中含有一个半胱氨酸残基，该残基协调催化锌离子。另一家族的 Metzincins，Astacins，没有半胱氨酸开关，因此通过其他方式维持潜伏状态。我们解决了来自欧洲小龙虾的原天蚕素 proastacin 的高分辨率晶体结构。它的前导肽段是报道的结构上最短的金属肽酶，具有独特的结构。它以与真正底物相反的方向贯穿活性部位裂隙。此外，一个保守的天冬氨酸，由前导肽段的宽环投射出来，与锌离子配位，而不是与成熟酶中发现的催化溶剂分子配位。激活通过两步有限蛋白水解发生，并需要柔性激活结构域的重大重排，该结构域变得刚性并形成底物结合裂隙的基础。成熟还需要新形成的 N 端被精确修剪，以便它可以参与埋藏的溶剂介导氢键网络，其中包括一个不变的活性部位残基。我们描述了一种新的潜伏和激活机制，它与其他金属肽酶和丝氨酸肽酶共享一些共同特征。
