Stopped flow fluorescence energy transfer measurement of the rate constants describing the reversible formation and the irreversible rearrangement of the elastase-alpha1-proteinase inhibitor complex.
Serpins are thought to inhibit proteinases by first forming a Michaelis-type complex that later converts into a stable inhibitory species. However, there is only circumstantial evidence for such a two-step reaction pathway. Here we directly observe the sequential appearance of two complexes by measuring the time-dependent change in fluorescence resonance energy transfer between fluorescein-elastase and rhodamine-alpha1-protease inhibitor. A moderately tight initial Michaelis-type complex EI1 (Ki = 0.38-0.52 microM) forms and dissociates rapidly (k1 = 1.5 x 10(6) M-1 s-1, k-1 = 0.58 s-1). EI1 then slowly converts into EI2 (k2 = 0.13 s-1), the fluorescence intensity of which is stable for at least 50 s. The two species differ by their donor-acceptor energy transfer efficiency (0. 41 and 0.26, respectively). EI2 might be the final product of the elastase + inhibitor association because its transfer efficiency is the same as that of a complex incubated for 30 min. The time-dependent change in fluorescence resonance energy transfer between fluorescein-elastase and rhodamine-eglin c, a canonical inhibitor, again allows the fast formation of a complex to be observed. However, this complex does not undergo any fluorescently detectable transformation.
Mellet
P
P
Laboratoire d'Enzymologie, INSERM U 392, CNRS URA 491, Université Louis Pasteur de Strasbourg, Strasbourg F-67000, France.
Boudier
C
C
Mely
Y
Y
Bieth
J G
JG
D000818:Animals; D004735:Energy Transfer; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D007700:Kinetics; D019272:Leukocyte Elastase / Q000737:chemistry* / Q000378:metabolism*; D008956:Models, Chemical; D010179:Pancreas / Q000201:enzymology; D011506:Proteins; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015843:Serpins / Q000378:metabolism / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D013552:Swine; D013997:Time Factors; D000515:alpha 1-Antitrypsin / Q000737:chemistry* / Q000378:metabolism*
D005456:Fluorescent Dyes; D011506:Proteins; D011994:Recombinant Proteins; D015843:Serpins; D000515:alpha 1-Antitrypsin; C037741:alpha 1-antitrypsin-leukocyte elastase complex; C028656:eglin proteinase inhibitors; D019272:Leukocyte Elastase; D016650:Fluorescein-5-isothiocyanate