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, a cylation, and translocation of the proteinase, To see whether acylation pre cedes or follows translocation, we have investigated the pH dependence of t he interaction of fluorescein isothiocyanate-elastase with rhodamine alpha( 1)-proteinase inhibitor (alpha(1)PI) using two independent methods: (i) kin etics of fluorescence energy transfer which yields k(2,f), the rate constan t for the fluorescently detected decay of the Michaelis-type complex (Melle t, P., Boudier, C., Mely, Y., and Bieth, J. G. (1998) J. Biol. Chem. 273, 9 119-9123); (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 elast ase. Both rate constants were found to be pH-independent and close to each other, indicating that acylation, a pH-dependent phenomenon, does not gover n the decay of the Michaelis-type complex and, therefore, follows transloca tion, 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 acyla tion is not a prerequisite for translocation, Moreover, the anhydro-elastas e-alpha(1)PI complex was found to be thermodynamically reversible, suggesti ng that translocation of active elastase might also be reversible. We propo se that serpins form a Michaelis-type complex EIM, which reversibly translo cates into EItr whose acylation yields the irreversible complex EIac. [GRAPHICS]