Allosteric modulation of the activity of thrombin

Substrates containing a P-3 aspartic residue are in general cleaved poorly by thrombin. This may be partly due to an unfavourable interaction between the P-3 aspartate and Glu(192) 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 P-3 aspartate. To investigate the importance of Glu(192) and exosite-binding in modulating thrombin's interactions with a P-3 aspartate, peptidyl chloromethanes based on the sequence of the thrombin receptor (containing a P-3 aspartate) have been synthesized and the kinetics of their inactivation of alpha-thrombin and the mutant Glu(192) --> Gln determined. The values of the inactivation rate constant (k(i)) for the chloromethanes containing a P-3 aspartate were about two-fold higher with the Glu(192) --> Gln mutant. A peptide based on the sequence of hirudin (rhir(52-65)), Which binds to the anion-binding exosite of thrombin, was an allosteric modulator of the amidolytic activity of the Glu(192) --> Gln mutant; a 5-fold decrease in the K-m value for the substrate D-Phe-pipecolyl-Arg-p-nitroanilide was observed in the presence of saturating concentrations of rhir-(52-65). This exosite-binding peptide also increased the k(i) values of chloromethanes containing a P-3 aspartate with both alpha-thrombin and the Glu(192) --> Gln mutant. However, the increases in the k(i) values were greater with the Glu(192) --> Gln mutant (5-fold compared with 2-fold for alpha-thrombin). Thus exosite binding does not seem to mitigate putative unfavourable interactions between Glu(192) and the P-3 aspartate. Moreover, increases in the k(i) caused by exosite binding were not unique to chloromethanes containing a P-3 aspartate; increases of the same magnitude were also observed when the P-3 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 S-2 and/or S-1 sites of thrombin.