The role of thrombin's Tyr-Pro-Pro-Trp motif in the interaction with fibrinogen, thrombomodulin, protein C, antithrombin III, and the Kunitz inhibitors.
When amino acids Pro60B, Pro60C, and Trp60D are deleted from thrombin, the resulting mutant (des-PPW) exhibits (compared to the wild-type enzyme): a similar second order rate constant of inhibition (k(on)) for diisopropyl fluorophosphate, and a comparable inhibition constant (K(i)) for benzamidine, suggesting that the charge stabilizing system and the primary binding pocket are little altered, if at all, by the mutation. As predicted from the x-ray structure, des-PPW is remarkably sensitive to the bovine pancreatic trypsin inhibitor, with a K(i) over 3 x 10(3) times tighter relative to thrombin, but des-PPW is also markedly less susceptible to inactivation by antithrombin III, with a k(on) that is over 100-fold lower. The catalytic constant (kcat) for most p-nitroanilide substrates tested is preserved or even increased, but the Michaelis constant (Km) increases. In contrast, the Km for the fibrinogen A alpha-chain is essentially unchanged, whereas kcat decreases approximately 50-fold. Unlike thrombin, the rate of fibrinopeptide B release becomes, following a lag phase, comparable to that of fibrinopeptide A. Inasmuch as des-PPW cleaves an additional peptide bond in the bovine fibrin alpha-chain, it remains a highly specific serine protease, which releases a single peptide from denatured casein (versus two with thrombin). Protein C activation by des-PPW is approximately 30 times slower than by thrombin in the absence, as well as in the presence, of calcium and thrombomodulin. Although this study confirms that the B-insertion restricts access to the active site cleft, it also suggests that other motifs and/or discrete amino acids are mainly responsible for the narrow specificity of thrombin.
Department of Haematology, University of Cambridge, Great Britain, Canada.
B F
BF
Le Bonniec
E R
ER
Guinto
R T
RT
MacGillivray
S R
SR
Stone
C T
CT
Esmon
D000595:Amino Acid Sequence; D000990:Antithrombin III / Q000378:metabolism / Q000494:pharmacology*; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology; D005340:Fibrinogen / Q000378:metabolism*; D005344:Fibrinopeptide A / Q000378:metabolism; D005345:Fibrinopeptide B / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011486:Protein C / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000378:metabolism*; D018179:Receptors, Thrombin; D013329:Structure-Activity Relationship; D013917:Thrombin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014360:Trypsin Inhibitor, Kunitz Soybean / Q000378:metabolism / Q000494:pharmacology*
D011486:Protein C; D011956:Receptors, Cell Surface; D018179:Receptors, Thrombin; D005344:Fibrinopeptide A; D005345:Fibrinopeptide B; D000990:Antithrombin III; D005340:Fibrinogen; D014360:Trypsin Inhibitor, Kunitz Soybean; D013917:Thrombin; D002118:Calcium