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 (Hir 52-65 ) and/or of heparin to these opposing exosites alters catalysis. We have investigated the contribution of subsites S 2 and S 3 to this allosteric transition by comparing the hydrolysis of two sets of fluorescence-quenched substrates having all natural amino acids at positions P 2 and P 3 . 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 Glu 192 participates in this modulation. We have examined the role of Glu 192 by comparing the catalytic activity of thrombin and its E192Q mutant. Mutation substantially diminishes the selectivity of thrombin. The substrate with the "best" P 2 residue was cleaved with a k cat /K m value only 49 times higher than the one having the "least favorable" P 2 residue (versus 636-fold with thrombin). Mutant E192Q also lost the strong preference of thrombin for positively charged P 3 residues and its strong aversion for negatively charged P 3 residues. Furthermore, both Hir 52-65 and heparin increased the k cat /K m value of substrate hydrolysis. We conclude that Glu 192 is critical for the P 2 and P 3 specificities of thrombin and for the allostery mediated through exosite 1.Thrombin (1, 2) is a multifunction serine protease finely tuned through: 1) restrictions fulfilled by the nature of the P 3 to P 3 Ј residues of the substrate, 1 that the S 3 to S 3 Ј subsites of the protease must accommodate, 2) steric hindrance resulting from surface loops, that limit access to the active site, 3) secondary exosites (one apolar and two positively charged) that strengthen the binding of cognate macromolecules (e.g. substrates such as fibrinogen but also inhibitors such as hirudin), and 4) allosteric control conferred by various cofactors (3). Several lines of evidence suggest that Glu 192 is a major player in the specificity of thrombin (4).2 Overall, hydrolysis by thrombin of its "best" substrates is little affected by the E192Q mutation, but new activities emerge that are severely restrained in normal thrombin. The E192Q mutant activates bovine factor X (5) and is efficiently inactivated by the serpin ␣ 1 -antitrypsin (6) and by the bovine pancreatic trypsin inhibitor (BPTI) 3 (7, 8). Glu 192 also seems to participate in the allosteric modulation of thrombin (4, 9). E192Q activates the anticoagulant protein C faster than thrombin; however, in the presence of the cofactor thrombomodulin, this difference disappears (4). Notably, the side chain of Glu 192 changes its conformation upon binding of a ligand to exosite 1 (10 -12).Among the effectors altering thrombin catalysis, molecules as diverse as inorganic ions, tryptamine analogs, polysaccharides, and proteins (or peptides derived from these proteins) have been identified. Sodium ions are allosteric modulators of thrombin, along with other serine proteases having a tyrosine in position...

Platelet Factor 4 (CXCL4) Seals Blood Clots by Altering the Structure of Fibrin

“…Similar effects were thus obtained with both reptilase and thrombin, supporting the hypothesis that PF4 interacted with fibrinogen and/or fibrin; alternately, PF4 would have to affect both proteases through discrete allosteric alterations. The rates of H-DPhe-Pro-Arg-pNA hydrolysis by either thrombin or reptilase were unaffected by large amounts of PF4 or poly-L-lysine and only marginally affected by Polybrene (data not shown), rendering it unlikely that the impact of PF4 on clot formation was mediated through allosteric alteration (39). That the effect of PF4 resulted from a specific interaction and not from an incompletely controlled parameter was also attested by the observation that a stoichiometric amount of an anti-PF4 antibody attenuated the effect of PF4 by 75% ( Fig.…”

Mapping of the Catalytic Groove Preferences of Factor Xa Reveals an Inadequate Selectivity for Its Macromolecule Substrates

“…Thrombin-In a previous study (29), we reported selectivity indexes of 636 and 20 in S 2 Ј for thrombin and trypsin, respectively. Accordingly, S 2 Ј of FXa would have a selectivity comparable with that of trypsin.…”

“…Therefore, three additional series of substrates were synthesized to fulfill the thrombin study; they had ABz-VGPRSFRDQ-EDDnp for a common framework in the P 2 and P 3 series and ABz-VGPRSFLDQ-EDDnp in the P 1 Ј series. Also, the values listed for thrombin concerning the P 2 Ј and P 3 Ј series are taken from Marque et al (29); they were obtained with fluorescence-quenched substrates having ABz-VGPRSFLLK(DNP)D for a common framework. Amino acids in P 3 , P 2 , P 1 Ј, P 2 Ј, or P 3 Ј that varied within each series are listed together with the estimated value of k cat /K m (in M Ϫ1 s Ϫ1 ) representing the weighted mean of at least three determinations (final standard errors were 20% or less).…”

“…Determination of the k cat /K m values of hydrolysis was performed essentially as described previously (16,29). Assuming that the reaction The five series of fluorescence-quenched substrates had ABz-VQFRSLGDQ-EDDnp for a common framework.…”

The Preparation of Fluorescence-Quenched Probes for Use in the Characterization of Human Factor Xa Substrate Binding Domains

“…Fluorescence quenched peptide substrates [6] spanning both sides of the scissile bond (P 1 -P 1 ') were used to investigate such P' specificities in a similar way to that used for other proteases [7,8]. These probes contain an N-terminal 2-aminobenzoyl (Abz) group and a penultimate 2,4-dinitrophenyl (Dnp) derivitized lysine (e.g.…”