Allosteric modulation of the activity of thrombin

Publisher Website
Google Scholar
Abstract
Substrates containing a P3 aspartic residue are in general cleaved poorly by thrombin. This may be partly due to an unfavourable interaction between the P3 aspartate and Glu192 in the active site of thrombin. In Protein C activation and perhaps also thrombin receptor cleavage, binding of ligands at the anion-binding exosite of thrombin seems to improve the activity of thrombin with substrates containing a P3 aspartate. To investigate the importance of Glu192 and exosite-binding in modulating thrombin's interactions with a P3 aspartate, peptidyl chloromethanes based on the sequence of the thrombin receptor (containing a P3 aspartate) have been synthesized and the kinetics of their inactivation of α-thrombin and the mutant Glu192 → Gln determined. The values of the inactivation rate constant (ki) for the chloromethanes containing a P3 aspartate were about two-fold higher with the Glu192 → Gln mutant. A peptide based on the sequence of hirudin (rhir52Ő65), which binds to the anion-binding exosite of thrombin, was an allosteric modulator of the amidolytic activity of the Glu192 → Gln mutant; a 5-fold decrease in the Km value for the substrate d-Phe-pipecolyl-Arg-p-nitroanilide was observed in the presence of saturating concentrations of rhir52Ő65. This exosite-binding peptide also increased the ki values of chloromethanes containing a P3 aspartate with both α-thrombin and the Glu192 → Gln mutant. However, the increases in the ki values were greater with the Glu192 → Gln mutant (5-fold compared with 2-fold for α-thrombin). Thus exosite binding does not seem to mitigate putative unfavourable interactions between Glu192 and the P3 aspartate. Moreover, increases in the ki caused by exosite binding were not unique to chloromethanes containing a P3 aspartate; increases of the same magnitude were also observed when the P3 position was occupied by the favourable d-phenylalanine in place of the unfavourable aspartate. The results obtained were consistent with exosite binding's causing changes in the conformation of the S2 and/or S1 sites of thrombin.