Hacisalihoglu Ayse, Panizzi Peter, Bock Paul E, Camire Rodney M, Krishnaswamy Sriram
Joseph Stokes Research Institute, Children's Hospital of Philadelphia, 3615 Civic Center Drive, Philadelphia, PA 19104, USA.
J Biol Chem. 2007 Nov 9;282(45):32974-82. doi: 10.1074/jbc.M706529200. Epub 2007 Sep 11.
The preferred pathway for prothrombin activation by prothrombinase involves initial cleavage at Arg(320) to produce meizothrombin, which is then cleaved at Arg(271) to liberate thrombin. Exosite binding drives substrate affinity and is independent of the bond being cleaved. The pathway for cleavage is determined by large differences in V(max) for cleavage at the two sites within intact prothrombin. By fluorescence binding studies in the absence of catalysis, we have assessed the ability of the individual cleavage sites to engage the active site of Xa within prothrombinase at equilibrium. Using a panel of recombinant cleavage site mutants, we show that in intact prothrombin, the Arg(320) site effectively engages the active site in a 1:1 interaction between substrate and enzyme. In contrast, the Arg(271) site binds to the active site poorly in an interaction that is approximately 600-fold weaker. Perceived substrate affinity is independent of active site engagement by either cleavage site. We further show that prior cleavage at the 320 site or the stabilization of the uncleaved zymogen in a proteinase-like state facilitates efficient docking of Arg(271) at the active site of prothrombinase. Therefore, we establish direct relationships between docking of either cleavage site at the active site of the catalyst, the V(max) for cleavage at that site, substrate conformation, and the resulting pathway for prothrombin cleavage. Exosite tethering of the substrate in either the zymogen or proteinase conformation dictates which cleavage site can engage the active site of the catalyst and enforces the sequential cleavage of prothrombin by prothrombinase.
凝血酶原酶激活凝血酶原的首选途径包括首先在精氨酸（320）处裂解产生中凝血酶，然后在精氨酸（271）处裂解以释放凝血酶。外结合位点驱动底物亲和力，且与被裂解的键无关。裂解途径由完整凝血酶原中两个位点的最大反应速度（Vmax）的巨大差异决定。通过在无催化情况下的荧光结合研究，我们评估了各个裂解位点在平衡状态下与凝血酶原酶中Xa活性位点结合的能力。使用一组重组裂解位点突变体，我们表明在完整的凝血酶原中，精氨酸（320）位点以底物与酶1:1的相互作用有效地结合活性位点。相比之下，精氨酸（271）位点与活性位点的结合较弱，其相互作用强度约低600倍。感知到的底物亲和力与任何一个裂解位点与活性位点的结合无关。我们进一步表明，在320位点预先裂解或使未裂解的酶原稳定在类似蛋白酶的状态下，有助于精氨酸（271）在凝血酶原酶活性位点的有效对接。因此，我们确定了任一裂解位点在催化剂活性位点的对接、该位点裂解的Vmax、底物构象以及凝血酶原裂解的最终途径之间的直接关系。底物在外结合位点的连接，无论是在酶原还是蛋白酶构象中，都决定了哪个裂解位点可以与催化剂的活性位点结合，并强制凝血酶原酶对凝血酶原进行顺序裂解。
