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Intrinsic fluorescence changes and rapid kinetics of proteinase deformation during serpin inhibition
Author(s) -
Tew Deborah J,
Bottomley Stephen P
Publication year - 2001
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(01)02305-5
Subject(s) - serpin , chemistry , kinetics , tryptophan , thrombin , biophysics , phenylalanine , fluorescence , conformational change , serine proteinase inhibitors , stereochemistry , biochemistry , enzyme , protease , biology , amino acid , serine protease , physics , platelet , quantum mechanics , immunology , gene
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 Using a variant of α 1 ‐antitrypsin in which both tryptophan residues have been replaced by phenylalanine, we have shown that the proteinase becomes partially unfolded during serpin inhibition.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.