Stopped flow fluorescence energy transfer measurement of the rate constants describing the reversible formation and the irreversible rearrangement of the elastase-α1-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-alpha(1)-protease inhibitor, A moderately tight initial Michaelis-type complex EI1 (K-i = 0.38-0.52 mu M) forms and dissociates rapidly (k(1) = 1.5 x 10(6) M-1 s(-1), k(-1) = 0.58 s(-1)). EI1 then slowly converts into EI2 (k(2) = 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.