Amide H/2H exchange reveals a mechanism of thrombin activation.
Thrombin is a dual action serine protease in the blood clotting cascade. Similar to other clotting factors, thrombin is mainly present in the blood in a zymogen form, prothrombin. Although the two cleavage events required to activate thrombin are well-known, little is known about why the thrombin precursors are inactive proteases. Although prothrombin is much larger than thrombin, prethrombin-2, which contains all of the same amino acids as thrombin, but has not yet been cleaved between Arg320 and Ile321, remains inactive. Crystal structures of both prethrombin-2 and thrombin are available and show almost no differences in the active site conformations. Slight differences were, however, seen in the loops surrounding the active site, which are larger in thrombin than in most other trypsin-like proteases, and have been shown to be important for substrate specificity. To explore whether the dynamics of the active site loops were different in the various zymogen forms of thrombin, we employed amide H/(2)H exchange experiments to compare the exchange rates of regions of thrombin with the same regions of prothrombin, prethrombin-2, and meizothrombin. Many of the surface loops showed less exchange in the zymogen forms, including the large loop corresponding to anion binding exosite 1. Conversely, the autolysis loop and sodium-binding site exchanged more readily in the zymogen forms. Prothrombin and prethrombin-2 gave nearly identical results while meizothrombin in some regions more closely resembled active thrombin. Thus, cleavage of the Arg320-Ile321 peptide bond is the key to formation of the active enzyme, which involves increased dynamics of the substrate-binding loops and decreased dynamics of the catalytic site.
Koeppe
Julia R
JR
Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093-0378, USA.
Komives
Elizabeth A
EA
D000577:Amides / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D041961:Deuterium Exchange Measurement; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000737:chemistry; D006801:Humans; D008969:Molecular Sequence Data; D010720:Phosphines; D011487:Protein Conformation / Q000187:drug effects; D011516:Prothrombin / Q000378:metabolism; D016415:Sequence Alignment; D013917:Thrombin / Q000737:chemistry / Q000378:metabolism*
D000577:Amides; D004792:Enzyme Precursors; D010720:Phosphines; D011516:Prothrombin; D013917:Thrombin; C080938:tris(2-carboxyethyl)phosphine; C026812:meizothrombin