Active site conformations of the thrombin mutant S195T free (A) or bound to PPACK (B,C) as determined by x-ray crystallography (). The side chain of T195 positions the methyl group toward an incoming substrate and the Oγ atom in the opposite direction but 3.1 Å away from the C42-C58 disulfide bond. This defective conformation of the nucleophile is corrected upon binding of PPACK (B, yellow sticks) that makes all the expected contacts with residues within the active site. The PPACK-bound conformation (C, PPACK removed for clarity) has an orientation of the Oγ atom of T195 compatible with substrate acylation. Shown are relevant H-bonding interactions. PPACK makes the expected contacts with the N atoms of T195 and G193 in the oxyanion hole, the N and O atoms of G216 and the carboxylate of D189, as well as a strong edge-to-face interaction with W215 in the aryl binding site. The presence of T195 does not alter the critical H-bonding interactions between D102 and H57, or the ion-pair between D194 and the N-terminus I16 that stabilizes the active site and oxyanion hole. There is no steric clash in the two orientations of T195 with residues within the active site. The distance of the methyl group from H57 is 4.1 Å in the free form (A) and 3.2 Å in the PPACK-bound form (C). Electron density (green mesh) is a 2F₀-F_c map contoured at 1.0 σ.

Distribution of the S195-H57 H-bonding distances (A) and dihedral angles (C-Cα-Cβ-Oγ) (B) of residue S195 in a total of 565 high resolution (<2.0 Å) structures of trypsin-like proteases and zymogens deposited in the PDB. The H-bonding distance between S195 and H57 features a Gaussian distribution with values of μ=3.1 Å and σ=0.3 Å barely affected by the presence of ligands in the active site (μ=3.0 Å, σ=0.3 Å). The S195 residue adopts a Gaussian distribution of dihedral angles with μ=156° and σ=37°, which is affected minimally by the presence of ligands within the active site (μ=148°, σ=36°). H-bonding distances and dihedral angles are also reported for the few zymogen strcutures documented in the PDB for the sake of completeness. The sample is however too small for statistical comparisons. (C) There is no correlation (r=0.51 or less) between the dihedral angle of S195 and its H-bonding distance from H57 in all three data sets.

(A) Rapid kinetic traces of FPR binding to thrombin mutant S195T in the 0–50 ms timescale. Shown are the traces obtained at 0 (red circles), 10 (cyan circles), 30 (green circles), 45 (magenta circles), and 60 (orange circles) μM FPR. Solid lines were drawn according to a single exponential function. Experimental conditions are: 0.1% PEG8000, 400 mM ChCl, 50 mM Tris, pH 8.0 at 15 °C. (B) Values of k_obs obtained from the analysis of the rapid kinetic traces according to a single exponential function. The solid horizontal line drawn at 400±10 s⁻¹ is the average of all k_obs values for FPR binding. The lack of dependence of k_obs on FPR concentration is unequivocal evidence of a pre-existing ensemble of inactive and active conformations with FPR selecting the active one for binding (). The value of k_obs=400±10 s⁻¹ is a measure of the rate of transition from the inactive to the active conformation. Because the kinetics refer to a pre-existing ensemble, this asymptotic value of k_obs also coincides with the k_off for FPR dissociation from the enzyme ().

(A) Effect of Na⁺ on the value of s=k_cat/K_m for the hydrolysis of FPR by thrombin wild-type (red circles) and S195T (blue circles). Under the effect of a cofactor, the value of s increases according to the linkage expression s=(s₀K_d+s₁x)/(K_d+x) (), where s₀ and s₁ are the asymptotic values of s at x=0 and x=∞, respectively, and K_d is the apparent equilibrium dissociation constant for the cofactor. The values of s are expressed in units of s₀ to facilitate comparison. In the case of wild-type, the value of s increases from s₀=3.8±0.1 μM⁻¹s⁻¹ to s₁=92±2 μM⁻¹s⁻¹. In the case of S195T, the activity drops drastically and the Na⁺ effect is practically abrogated (s₀=1.8±0.1 mM⁻¹s⁻¹ and s₁=2.3±0.1 mM⁻¹s⁻¹). Experimental conditions are: 0.1% PEG8000, 5 mM Tris, pH 8.0 at 25 °C. (B) Na+ binding curve for wild-type (red circles) and S195T (blue circles) obtained by titration of the intrinsic fluorescence of the protein. The total change in fluorescence was 11% for wild-type and 13% for S195T. Mutation of S195 has little effect on the value of the equilibrium dissociation constant K_d that changes from 8.2±0.8 mM in the wild-type to 6.2±0.2 mM in the mutant. Experimental conditions are: 0.1% PEG8000, 50 mM Tris, pH 8.0 at 15 °C. (C) Effect of thrombomodulin on the value of s=k_cat/K_m for the hydrolysis of protein C by thrombin wild-type (red circles) and S195T (blue circles). The values of s are expressed in units of s₀ as in panel (A) to facilitate comparison. In the case of wild-type (red circles), the value of s increases 2,100-fold from s₀=0.15±0.01 mM⁻¹s⁻¹ to s₁=310±10 mM⁻¹s⁻¹. In the case of S195T (blue circles), the activity drops drastically and the effect of thrombomodulin is significantly reduced (s₀=0.0020±0.0001 mM⁻¹s⁻¹ and s₁=0.36±0.02 mM⁻¹s⁻¹). The value of K_d=2.7±0.1 nM for thrombomodulin binding to wild-type is practically identical to the value of K_d=2.6±0.1 nM for the S195T mutant. Experimental conditions are: 0.1% PEG8000, 145 mM NaCl, 5 mM CaCl₂, 5 mM Tris, pH 7.4 at 37 °C.

Effect of Na⁺ on the value of s=k_cat/K_m for the hydrolysis of a chromogenic substrate by wild-type (red) and mutant S195T (blue) of activated protein C (A) and clotting factor Xa (B). The values of s are expressed in units of s₀ as in to facilitate comparison. As observed for thrombin (), the value of s in the wild-type increases from s₀=2.1±0.1 mM⁻¹s⁻¹ to s₁=69±2 mM⁻¹s⁻¹ for activated protein C (A) and from s₀=1.4±0.1 μM⁻¹s⁻¹ to s₁=28±1 μM⁻¹s⁻¹ for factor Xa (B). In the case of the S195T mutant, activity drops significantly and the Na⁺ effect is practically abrogated (s₀=0.11±0.01 mM⁻¹s⁻¹ and s₁=0.20±0.01 mM⁻¹s⁻¹ for activated protein C; s₀=18±1 mM⁻¹s⁻¹, s₁=38±1 mM⁻¹s⁻¹ for factor Xa). Experimental conditions are: (A) 0.1% PEG8000, 5 mM EDTA, 5 mM Tris, pH 8.0 at 25 °C; (B) 0.1% PEG8000, 5 mM EDTA, 50 mM HEPES, pH 8.0 at 25°C.