title: Intrinsic fluorescence changes and rapid kinetics of proteinase deformation during serpin inhibition.
abstract: 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 Using a variant of alpha(1)-antitrypsin in which both tryptophan residues have been replaced by phenylalanine, we have shown that the proteinase becomes partially unfolded during serpin inhibition.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, alpha(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 2.8x10(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.
authors: Department of Biochemistry and Molecular Biology, P.O. Box 13D, Monash University, 3800, Clayton, Vic., Australia.
authors: D J
authors: DJ
authors: Tew
authors: S P
authors: SP
authors: Bottomley
mesh_terms: D007700:Kinetics; D010649:Phenylalanine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000378:metabolism*; D015843:Serpins / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods; D013329:Structure-Activity Relationship; D013997:Time Factors; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000515:alpha 1-Antitrypsin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D015842:Serine Proteinase Inhibitors; D015843:Serpins; D000515:alpha 1-Antitrypsin; D010649:Phenylalanine; D014364:Tryptophan