The role of Glu192 in the allosteric control of the S2' and S3' subsites of thrombin

Abstract

Thrombin is an allosteric protease controlled through exosites flanking the catalytic groove. Binding of a peptide derived from hirudin (Hir52-65) and /or of heparin to these opposing exosites alters catalysis. We have investigated the contribution of subsites S2' and S3' to this allosteric transition by comparing the hydrolysis of two sets of fluorescence-quenched substrates having all natural amino acids at positions P2' and P3'. Regardless of the amino acids, Hir52-65 decreased, and heparin increased the k(cat)/K(m) value of hydrolysis by thrombin. Several lines of evidence have suggested that Glu192 participates in this modulation. We have examined the role of Glu192 by comparing the catalytic activity of thrombin and its E192Q mutant. Mutation substantially diminishes the selectivity of thrombin. The substrate with the 'best' P2' residue was cleaved with a k(cat)/K(m) value only 49 times higher than the one having the 'least favorable' P2' residue (versus 636-fold with thrombin). Mutant E192Q also lost the strong preference of thrombin for positively charged P3' residues and its strong aversion for negatively charged P3' residues. Furthermore, both Hir52-65 and heparin increased the k(cat)/K(m) value of substrates hydrolysis. We conclude that Glu192 is critical for the P2' and P3' specificities of thrombin and for the allostery mediated through exosite 1.