Apparent Formation of Sodium Dodecyl Sulfate-stable Complexes between Serpins and 3,4-Dichloroisocoumarin-inactivated Proteinases Is Due to Regeneration of Active Proteinase from the Inactivated Enzyme

Abstract

Protein proteinase inhibitors of the serpin family were recently reported to form SDS-stable complexes with inactive serine proteinases modified at the catalytic serine with 3,4-dichloroisocoumarin (DCI) that resembled the complexes formed with the active enzymes (Christensen, S., Valnickova, Z., Thøgersen, I. B., Pizzo, S. V., Nielsen, H. R., Roepstorff, P., and Enghild, J. J. (1995) J. Biol. Chem. 270, 14859 -14862). The discordance between these findings and other reports that similar active site modifications of serine proteinases block the ability of serpins to form SDS-stable complexes prompted us to investigate the mechanism of complex formation between serpins and DCI-inactivated enzymes. Both neutrophil elastase and ␤-trypsin inactivated by DCI appeared to form SDS-stable complexes with the serpin, ␣ 1 -proteinase inhibitor (␣ 1 PI), as reported previously. However, several observations suggested that such complex formation resulted from a reaction not with the DCI enzyme but rather with active enzyme regenerated from the DCI enzyme by a ratelimiting hydrolysis reaction. Thus (i) complex formation was blocked by active site-directed peptide chloromethyl ketone inhibitors; (ii) the kinetics of complex formation indicated that the reaction was not second order but rather showed a first-order dependence on DCI enzyme concentration and zero-order dependence on inhibitor concentration; and (iii) complex formation was accompanied by stoichiometric release of a peptide having the sequence SIPPE corresponding to cleavage at the ␣ 1 PI reactive center P1-P1 bond. Quantitation of kinetic constants for DCI and ␣ 1 PI inactivation of human neutrophil elastase and trypsin and for reactivation of the DCI enzymes showed that the observed complex formation could be fully accounted for by ␣ 1 PI preferentially reacting with active enzyme regenerated from DCI enzyme during the reaction. These results support previous findings of the critical importance of the proteinase catalytic serine in the formation of SDS-stable serpin-proteinase complexes and are in accord with an inhibitory mechanism in which the proteinase is trapped at the acyl intermediate stage of proteolysis of the serpin as a substrate.

The Clearance of Thrombin-antithrombin and Related Serpin-enzyme Complexes from the Circulation: Role of Various Hepatocyte Receptors

“…Enghild et al (24) thus asked whether the formation of such complexes was sufficient to accelerate clearance, and found that complexes of serpin and 3,4-dichloroisocoumarin (DCI)-inactivated serine proteinase were cleared from the murine circulation more rapidly than the corresponding native serpin. However, further studies indicated that the DCIproteinase complex decayed, releasing active proteinase which then could covalently complex with the serpin (25), effectively converting the encounter complex into an SEC. Thus, the results of in vivo clearance studies suggest that maximal acceleration of serpin clearance requires formation of covalently associated, denaturation-resistant, SEC.…”

The Serpin Inhibitory Mechanism Is Critically Dependent on the Length of the Reactive Center Loop

“…V 0 is the rate of change in A 405 at time zero, k off is the first-order rate constant for complex dissociation, [E Ϫ I] 0 is the starting concentration of complex, and TN is the turnover number for the hydrolysis of substrate by enzyme under the conditions of the experiment and was independently measured. Complex dissociation is irreversible because serpin is released from the complex in an inactive cleaved form rather than in the native intact form (24). The dissociation rate of the ␣ 1 -antitrypsin Add-2-factor Xa complex was too fast to be analyzed by the above method and was therefore determined by SDS-PAGE.…”

The pH Dependence of Serpin-Proteinase Complex Dissociation Reveals a Mechanism of Complex Stabilization Involving Inactive and Active Conformational States of the Proteinase Which Are Perturbable by Calcium

“…Thrombin, factor Xa and bovine trypsin were active site titrated and found to be 70 -100% active based on comparisons with the concentrations measured from the 280-nm absorbance and published absorption coefficients (4,39). The concentrations of HNE and porcine trypsin were based on a stoichiometric titration of the enzyme with ␣ 1 PI as described (40). Proteinase concentrations were routinely determined from initial rates of proteinase hydrolysis, monitored from the absorbance change at 405 nm, of the substrates, S-2238 (Chromogenix) for thrombin, Spectrozyme FXa (American Diagnostica) for factor Xa, S-2222 (Chromogenix) for trypsin, or methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide (Sigma) for HNE under standard conditions after calibrating these rates using known concentrations of active site titrated proteinase.…”