Tine variable region-1 from tissue-type plasminogen activator confers specificity for plasminogen activator inhibitor-1 to thrombin by facilitating catalysis: Release of a kinetic block by a heterologous protein surface loop

Substitution of the native variable region-1 (VR1/37-loop) of thrombin by t he corresponding VR1 of tissue-type plasminogen activator (thrombin-VR1(tPA )) increases the rate of inhibition by plasminogen activator inhibitor type 1 (PAI-1) by three orders of magnitude, and is thus sufficient to confer P AI-1 specificity to a heterologous serine protease. A structural and kineti cal approach to establish the function of the VR1 loop of t-PA in the conte xt of the thrombin-VR1(tPA) variant is described. The crystal structure of thrombin-VR1(tPA) was resolved and showed a conserved overall alpha-thrombi n structure, but a partially disordered VR1 loop as also reported for t-PA. The contribution of a prominent charge substitution close to the active si te was studied using charge neutralization variants thrombin-E39Q(c39) and thrombin-VR1(tPA)-R304Q(c39), resulting in only fourfold changes in the PAI -1 inhibition rate. Surface plasmon resonance revealed that the affinity of initial reversible complex formation between PAI-1 and catalytically inact ive Ser195 --> Ala variants of thrombin and thrombin-VR1(tPA) is only incre ased fivefold, i.e. K-D is 652 and 128 nM for thrombin-S195A and thrombin-S 195A-VR1(tPA), respectively. We established that the partition ratio of the suicide substrate: reaction between the proteases and PAI-1 was largely un affected in any variant Studied. Hirugen allosterically decreases the rate of thrombin inhibition by PAI-1 2.5-fold and of thrombin-VR1(tPA) 20-fold, by interfering with a unimolecular step in the reaction, not by decreasing initial complex formation or by altering the stoichiometry. Finally, kineti c modeling demonstrated that acylation is the rate-limiting step in thrombi n inhibition by PAI-1 (k similar to 10(-3) s(-1)) and this kinetic block is alleviated by the introduction of the tPA-VR1 into thrombin (k > 1 s(-1)). We propose that the length, flexibility and different charge architecture of the VR1 loop of t-PA invoke an induced fit of the reactive center loop o f PAI-1, thereby enhancing the rate of:acylation in the Michaelis complex b etween thrombin-VR1(tPA) and PAI-1 by more than two orders of magnitude. (C ) 1999 Academic Press.