| Abstract
| - The conversion of the reactive center bond of the serpin α1-proteinase inhibitor (α1-PI, alsoknown as α1-antitrypsin) from Met-Ser to Arg-Ser decreases the rate at which it inhibits neutrophil elastaseand endows it with the ability to inhibit thrombin and activated protein C (APC). Another serpin, heparincofactor II (HCII), contains a unique N-terminal extension that binds thrombin exosite 1. We fused residues1−75 of HCII to the N-terminus of α1-PI M358R, forming an HCII−α1-PI chimera (HAPI M358R). Itinhibited α-thrombin 21-fold faster than α1-PI M358R, with second-order rate constants of 2.3 × 108M-1 min-1 versus 1.1 × 107 M-1 min-1, respectively. When γT-thrombin, which lacks an intact exosite1, was substituted for α-thrombin, the kinetic advantage of HAPI M358R over α1-PI M358R was reducedto 9-fold, whereas APC and trypsin, proteases lacking exosite 1-like regions, were inhibited only 1.3- and2-fold more rapidly by HAPI M358R than by α1-PI M358R, respectively. Maximal enhancement of α1-PI M358R activity required the acidic residues found between HCII residues 55 and 75, because noenhancement was observed either by fusion of residues 1−54 alone or by fusion of a mutated HCII acidicextension in which all Glu and Asp residues between positions 55 and 75 were neutralized by mutation.Fusing residues 55−75 to α1-PI M358R yielded a relative rate enhancement of only 6-fold, suggesting aneed for the full tail region to achieve maximal enhancement. Our results suggest that transfer of theN-terminal acidic extension of HCII to α1-PI M358R enhanced its inhibition of thrombin by conferringthe ability to bind exosite 1 on HAPI M358R. This enhancement may aid in efforts to tailor this inhibitorfor therapeutic use.
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