Contribution of Lysine 60f to S1‘ Specificity of Thrombin

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Abstract
Lys60f has been proposed to limit the S1‘ substrate binding site specificity of thrombin to small polar P1‘ residues by occluding the S1‘ binding pocket, based on the X-ray crystal structure of thrombin. To test this proposal, we prepared a Lys → Ala (K60fA) mutant of recombinant thrombin and determined whether this mutation enhanced the reactivity of thrombin with a variant inhibitor [antithrombin (AT)-Denver] and a substrate (protein C) containing poorly recognized P1‘ Leu residues. AT-Denver in the presence of heparin inhibited K60fA thrombin with a second-order association rate constant [k = (4.2 ± 0.1) × 105 M-1 s-1] that was 3.2-fold faster than thrombin [k = (1.3 ± 0.1) × 105 M-1 s-1]. Wild-type AT (P1‘ Ser) under the same conditions inhibited K60fA thrombin with a 2.5-fold slower rate constant [k = (1.1 ± 0.1) × 107 M-1 s-1] than thrombin [k = (2.8 ± 0.1) × 107 M-1 s-1]. These results indicate an overall 8.3-fold improvement in the recognition of the P1‘ Leu of AT-Denver by K60fA thrombin over that of wild-type thrombin; i.e., the K60fA mutation partly overcomes the defect in thrombin inhibition produced by the P1‘ mutation in AT-Denver. Resolution of the two-step reactions of AT and AT-Denver with wild-type and mutant thrombins revealed that the enhanced recognition of P1‘ Leu in AT-Denver by K60fA thrombin occurs primarily in the second reaction step in which a noncovalent AT−thrombin encounter complex is converted to a stable, covalent complex. Thrombin K60fA activated Gla-domainless protein C (GDPC) ∼2- and ∼4-fold faster than thrombin in the presence and absence of thrombomodulin (TM), respectively, consistent with an improved interaction of the Leu P1‘ residue with the mutant S1‘ pocket. In contrast, the mutant thrombin clotted fibrinogen (P1‘ Gly) ∼3-fold slower than thrombin. Kinetic analysis revealed that the improvement in the catalytic rate of activation of GDPC by K60fA thrombin in the presence of TM was localized in the second reaction step, as reflected by an ∼2-fold increase in kcat. Direct binding studies showed that the K60fA mutation minimally affected the affinity of thrombin for Na+, indicating that the changes in S1‘ site-specificity of K60fA thrombin did not result from altering the allosteric transition induced by Na+. We conclude that Lys60f limits the P1‘ substrate and inhibitor specificity of thrombin by influencing the size and polarity of the S1‘ site which thereby affects the stability of the transition state for cleavage of the scissile bond in the second reaction step.