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

FEBS Letters

Tew, Deborah J¹  Bottomley, Stephen P¹ 

[1] Department of Biochemistry and Molecular Biology, P.O. Box 13D, Monash University, 3800 Clayton, Vic., Australia

关键词: Serpin;    Proteinase;    Protein folding;    Conformational change;    α1-AT;    α1-antitrypsin;    EI*;    final serpin proteinase complex;    EIM;    initial Michaelis-type complex;    λ ex;    excitation wavelength;    λ max;    emission maximum;    serpin;    serine proteinase inhibitor;   

DOI  :  10.1016/S0014-5793(01)02305-5

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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 Using a variant of α₁-antitrypsin in which both tryptophan residues have been replaced by phenylalanine, we have shown that the proteinase becomes partially unfolded during serpin inhibition.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, α₁-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 2.8×10⁶ 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.

【 授权许可】

Unknown