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J Biol Chem, Vol. 275, Issue 2, 809-816, January 14, 2000

The Role of Glu¹⁹² in the Allosteric Control of the S₂' and S₃' Subsites of Thrombin^*

Pierre-Emmanuel Marque, Roberta Spuntarelli, Luiz Juliano^§, Martine Aiach, and Bernard F. Le Bonniec^¶

From the  INSERM, U428, Université Paris V, Faculté de Pharmacie 4 Avenue de l'Observatoire, 75270 Paris Cedex 06, France and the ^§ Departamento de Biofisica, Escola Paulista de Medicina, Rua Três de Maio, 100, 04044-020 São Paulo, SP, Brazil

Thrombin is an allosteric protease controlled through exosites flanking the catalytic groove. Binding of a peptide derived from hirudin (Hir^52-65) 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, Hir^52-65 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 Hir^52-65 and heparin increased the k_cat/K_m value of substrate hydrolysis. We conclude that Glu¹⁹² is critical for the P₂' and P₃' specificities of thrombin and for the allostery mediated through exosite 1.

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