Intrinsic fluorescence changes and rapid kinetics of proteinase deformation during serpin inhibition

The X‐ray crystal structure of the serpin–proteinase complex suggested that the serpin deformed the proteinase thereby inactivating the molecule. Using a variant of α 1 ‐antitrypsin in which both tryptophan residues have been replaced by phenylalanine, we have shown that the proteinase becomes partially unfolded during serpin inhibition. The tryptophan free variant, α 1 ‐antitrypsin (FF) , is fully active as an inhibitor of thrombin. Thrombin has a fluorescence emission maximum of 340 nm which blue shifts to 346 nm, concomitant with a 40% increase in intensity, upon formation of the serpin–proteinase complex indicative of substantial conformational change within the proteinase. Stopped‐flow analysis of the fluorescence changes within the proteinase indicated a two‐step mechanism. A fast bimolecular reaction with a rate constant of 2.8×10 6 M −1 s −1 is followed by a slow unimolecular process with a rate of 0.26 s −1 that is independent of concentration. We propose that the first rate is formation of an initial complex which is then followed by a slower process involving the partial unfolding of the proteinase during its translocation to the opposite pole of the serpin.