Na⁺ binding to meizothrombin desF1

• Published in: Cellular and molecular life sciences : CMLS 2008-11, Vol.65 (22), p.3688-3697
• Main Authors: Papaconstantinou, M. E, Gandhi, P. S, Chen, Z, Bah, A, Di Cera, E
• Format: Article
• Language: English
• Subjects: allostery; Amino Acid Chloromethyl Ketones; Biochemistry; Biomedical and Life Sciences; Biomedicine; Cell Biology; Crystal structure; Crystallography, X-Ray; Enzyme Activation - drug effects; Enzyme Precursors - chemistry; Enzyme Precursors - metabolism; Enzymes; Flow measurement; Humans; Kinetics; Life Sciences; linkage; meizothrombin; Models, Molecular; Molecular biology; Na⁺ binding; Protein Conformation; Research Article; Sodium - metabolism; Sodium - pharmacology; thrombin; Thrombin - chemistry; Thrombin - metabolism
• ISSN: 1420-682X; 1420-9071; 1420-9071
• DOI: 10.1007/s00018-008-8502-7

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.