Na⁺ binding to meizothrombin desF1

Abstract.

Meizothrombin is the physiologically active intermediate generated by a single cleavage of prothrombin at R320 to separate the A and B chains. Recent evidence has suggested that meizothrombin, like thrombin, is a Na⁺-activated enzyme. In this study we present the first X-ray crystal structure of human meizothrombin desF1 solved in the presence of the active site inhibitor PPACK at 2.1 Å resolution. The structure reveals a Na⁺ binding site whose architecture is practically identical to that of human thrombin. Stopped-flow measurements of Na⁺ binding to meizothrombin desF1 document a slow phase of fluorescence change with a k _obs decreasing hyperbolically with increasing [Na⁺], consistent with the existence of three conformations in equilibrium, E*, E and E:Na⁺, as for human thrombin. Evidence that meizothrombin exists in multiple conformations provides valuable new information for studies of the mechanism of prothrombin activation.