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.
Papaconstantinou
M E
ME
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110, USA.
Gandhi
P S
PS
Chen
Z
Z
Bah
A
A
Di Cera
E
E
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