Evidence that translocation of the proteinase precedes its acylation in the serpin inhibition pathway.

The inhibition of proteinases by serpins involves cleavage of the serpin, acylation, and translocation of the proteinase. To see whether acylation precedes or follows translocation, we have investigated the pH dependence of the interaction of fluorescein isothiocyanate-elastase with rhodamine alpha(1)-proteinase inhibitor (alpha(1)PI) using two independent methods: (i) kinetics of fluorescence energy transfer which yields k(2,f), the rate constant for the fluorescently detected decay of the Michaelis-type complex (Mellet, P., Boudier, C., Mély, Y., and Bieth, J. G. (1998) J. Biol. Chem. 273, 9119-9123); (ii) kinetics of elastase-catalyzed hydrolysis of a substrate in the presence of alpha(1)PI, which yields k(2,e), the rate constant for the conversion of the Michaelis-type complex into irreversibly inhibited elastase. Both rate constants were found to be pH-independent and close to each other, indicating that acylation, a pH-dependent phenomenon, does not govern the decay of the Michaelis-type complex and, therefore, follows translocation. On the other hand, anhydro-elastase reacts with alpha(1)PI to form a Michaelis-type complex that translocates into a second complex with a rate constant close to that measured with active elastase, confirming that acylation is not a prerequisite for translocation. Moreover, the anhydro-elastase-alpha(1)PI complex was found to be thermodynamically reversible, suggesting that translocation of active elastase might also be reversible. We propose that serpins form a Michaelis-type complex EI(M), which reversibly translocates into EI(tr) whose acylation yields the irreversible complex EI(ac). [see text]