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.
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., Thogersen, 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 beta-trypsin inactivated by DCI appeared to form SDS-stable complexes with the serpin, alpha1-proteinase inhibitor (alpha1PI), 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 rate-limiting 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 alpha1PI reactive center P1-P1' bond. Quantitation of kinetic constants for DCI and alpha1PI 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 alpha1PI 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.
Olson
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Center for Molecular Biology of Oral Diseases, University of Illinois, Chicago, Illinois 60612-7213, USA.
Swanson
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Patston
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Björk
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D000818:Animals; D002417:Cattle; D003374:Coumarins / Q000494:pharmacology*; D010450:Endopeptidases / Q000737:chemistry* / Q000378:metabolism; D004789:Enzyme Activation; D006801:Humans; D049934:Isocoumarins; D015842:Serine Proteinase Inhibitors / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D015843:Serpins / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry*
D003374:Coumarins; D049934:Isocoumarins; D015842:Serine Proteinase Inhibitors; D015843:Serpins; D012967:Sodium Dodecyl Sulfate; C046235:3,4-dichloroisocoumarin; D010450:Endopeptidases