Fischer B E, Schlokat U, Himmelspach M, Dorner F
Biomedical Research Center, Immuno AG, Orth an der Donau, Austria.
Protein Eng. 1998 Aug;11(8):715-21. doi: 10.1093/protein/11.8.715.
Prothrombin (coagulation factor II) is the inactive precursor molecule of thrombin (coagulation factor IIa). Proteolytic cleavage of the peptide bond Arg320-Ile321 converts prothrombin into the two-chain thrombin precursor meizothrombin. Meizothrombin hydrolyses peptidyl substrates, but cleavage of fibrinogen is poor. Unfortunately, meizothrombin exhibits a significant autocatalytic activity and thus is not structurally stable in solution. Hirudin, the 65-residue peptide anticoagulant from the salivary gland of the European leech Hirudo medicinalis, is a highly specific and effective thrombin inhibitor. To study the interactions of meizothrombin and hirudin, recombinant prothrombin with active site Asp419 replaced by Asn (D419N-prothrombin) was produced in CHO cells and transformed into D419N-meizothrombin in vitro. D419N-meizothrombin exhibited no proteolytic and autocatalytic activity. D419N-meizothrombin was affinity purified at an immobilized C-terminal hirudin-derived peptide demonstrating the presence and activity of the anion binding exosite. D419N-meizothrombin exhibited binding activity to hirudin immobilized at the solid phase in an ELISA. Incubation of D419N-meizothrombin with hirudin resulted in a significant increase of intrinsic fluorescence. Fluorescence titration of D419N-meizothrombin with hirudin produced a sharp break in the titration curve at the molar equivalence point and a total fluorescence enhancement of 24%. However, the titration curve did not reflect a simple binding mechanism. Incubation of D419N-meizothrombin with fibrinopeptide A and C-terminal hirudin peptide 54-65 did not change fluorescence emission. Trp468 located in the gamma-loop of thrombin was replaced by Phe in the double-mutant D419N/W468F-thrombin. Similar to D419N-thrombin and D419N-meizothrombin, formation of the D419N/W468F-thrombin/hirudin complex resulted a significant increase in intrinsic fluorescence. Apparently, the binding of hirudin induces similar structural changes in both meizothrombin and thrombin. The structural change does not involve the flexible gamma-loop. The results suggest that meizothrombin binds hirudin similar to thrombin.
凝血酶原（凝血因子II）是凝血酶（凝血因子IIa）的无活性前体分子。肽键Arg320-Ile321的蛋白水解切割将凝血酶原转化为双链凝血酶前体中凝血酶。中凝血酶能水解肽基底物，但对纤维蛋白原的切割效果较差。不幸的是，中凝血酶表现出显著的自催化活性，因此在溶液中结构不稳定。水蛭素是欧洲医用水蛭唾液腺分泌的一种由65个氨基酸残基组成的肽类抗凝剂，是一种高度特异性且有效的凝血酶抑制剂。为了研究中凝血酶与水蛭素的相互作用，在CHO细胞中产生了活性位点Asp419被Asn取代的重组凝血酶原（D419N-凝血酶原），并在体外将其转化为D419N-中凝血酶。D419N-中凝血酶没有蛋白水解和自催化活性。D419N-中凝血酶通过固定化的C端水蛭素衍生肽进行亲和纯化，证明了阴离子结合外位点的存在和活性。在酶联免疫吸附测定（ELISA）中，D419N-中凝血酶对固定在固相上的水蛭素表现出结合活性。D419N-中凝血酶与水蛭素孵育导致固有荧光显著增加。用水蛭素对D419N-中凝血酶进行荧光滴定，在摩尔当量点处滴定曲线出现急剧转折，总荧光增强24%。然而，滴定曲线并不反映简单的结合机制。D419N-中凝血酶与纤维蛋白肽A和C端水蛭素肽54-65孵育不会改变荧光发射。位于凝血酶γ-环中的Trp468在双突变体D419N/W468F-凝血酶中被Phe取代。与D419N-凝血酶和D419N-中凝血酶类似，D419N/W468F-凝血酶/水蛭素复合物的形成导致固有荧光显著增加。显然，水蛭素的结合在中凝血酶和凝血酶中诱导了类似的结构变化。这种结构变化不涉及灵活的γ-环。结果表明，中凝血酶与水蛭素的结合方式与凝血酶类似。
