Thrombin-fibrinogen interaction: pH dependence and effects of the slow-->fast transition.
A recently developed strategy capable of measuring the equilibrium dissociation constant for thrombin-fibrinogen interaction has been used to explore the pH dependence of the interaction and the effects of thrombin conformational transitions. The dependence of fibrinogen binding to thrombin in the pH range 6-10 is bell-shaped and remarkably similar to that obtained in the case of the small synthetic amide substrate tosyl-Gly-Pro-Arg-p-nitroanilide-AcOH. Since the synthetic substrate contains no groups that can ionize in the pH range 6-10, the bell-shaped curve must reflect ionization reactions of two groups of the enzyme with pK1 = 7.53 +/- 0.09 and pK2 = 8.80 +/- 0.09. These groups can be identified as the catalytic histidine, His57, and the amino terminus of the B chain, Ile16, respectively. Deprotonation of His57 in the acidic region is important for optimal binding, while protonation of Ile16 in the alkaline region is critical for the formation of a salt bridge with Asp194, which guarantees the conformational stability of the enzyme. The loss of binding free energy at low (< 7.0) and high (> 9.0) pH values is linked to protonation of His57 and deprotonation of Ile16, respectively. The first 51 residues of the A alpha chain of fibrinogen are known to be necessary and sufficient for optimal recognition by thrombin, but none of them contributes to the pH dependence of fibrinogen binding in the pH range examined. Hence, the two possible ionizable groups of the A alpha chain, i.e., the amino terminus Alal and His24, make no contacts with the thrombin surface.(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110.
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A
Mathur
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Schlapkohl
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Di Cera
D000215:Acylation; D000595:Amino Acid Sequence; D002863:Chromogenic Compounds / Q000378:metabolism; D005340:Fibrinogen / Q000378:metabolism*; D005344:Fibrinopeptide A / Q000378:metabolism; D006639:Histidine / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007532:Isoleucine / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism; D011522:Protons; D013917:Thrombin / Q000378:metabolism*
D002863:Chromogenic Compounds; D009842:Oligopeptides; D011522:Protons; D007532:Isoleucine; D005344:Fibrinopeptide A; D006639:Histidine; C014413:chromozym TH; D005340:Fibrinogen; D013917:Thrombin