Na+ binding to meizothrombin desF1.
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 A 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.
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110, USA.
M E
ME
Papaconstantinou
P S
PS
Gandhi
Z
Z
Chen
A
A
Bah
E
E
Di Cera
D000590:Amino Acid Chloromethyl Ketones; D018360:Crystallography, X-Ray; D004789:Enzyme Activation / Q000187:drug effects; D004792:Enzyme Precursors / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D011487:Protein Conformation; D012964:Sodium / Q000378:metabolism* / Q000494:pharmacology; D013917:Thrombin / Q000737:chemistry* / Q000378:metabolism*
D000590:Amino Acid Chloromethyl Ketones; D004792:Enzyme Precursors; D012964:Sodium; D013917:Thrombin; C026812:meizothrombin; C040308:phenylalanyl-prolyl-arginine-chloromethyl ketone