Portal de Periódicos da CAPES

Role of the Catalytic Serine in the Interactions of Serine Proteinases with Protein Inhibitors of the Serpin Family

1995; Elsevier BV; Volume: 270; Issue: 50 Linguagem: Inglês

10.1074/jbc.270.50.30007

1083-351X

Steven T. Olson, Paul Bock, Jan KVASSMAN, Joseph D. Shore, Daniel A. Lawrence, David Ginsburg, Ingemar Björk,

Coagulation, Bradykinin, Polyphosphates, and Angioedema

The contribution of a covalent bond to the stability of complexes of serine proteinases with inhibitors of the serpin family was evaluated by comparing the affinities of β-trypsin and the catalytic serine-modified derivative, β-anhydrotrypsin, for several serpin and non-serpin (Kunitz) inhibitors. Kinetic analyses showed that anhydrotrypsin had little or no ability to compete with trypsin for binding to α1-proteinase inhibitor (α1PI), plasminogen activator inhibitor 1 (PAI-1), antithrombin (AT), or AT-heparin complex when present at up to a 100-fold molar excess over trypsin. By contrast, equimolar levels of anhydrotrypsin blocked trypsin binding to non-serpin inhibitors. Equilibrium binding studies of inhibitor-enzyme interactions monitored by inhibitor displacement of the fluorescence probe, p-aminobenzamidine, from the enzyme active site, confirmed that the binding of serpins to anhydrotrypsin was undetectable in the case of α1PI or AT (KI > 10−5M), of low affinity in the case of AT-heparin complex (KI 7-9 × 10−6M), and of moderate affinity in the case of PAI-1 (KI 2 × 10−7M). This contrasted with the stoichiometric high affinity binding of the serpins to trypsin as well as of the non-serpin inhibitors to both trypsin and anhydrotrypsin. Maximal KI values for serpin-trypsin interactions of 1 to 8 × 10−11M, obtained from kinetic analyses of association and dissociation rate constants, indicated that the affinity of serpins for trypsin was minimally 4 to 6 orders of magnitude greater than that of anhydrotrypsin. Anhydrotrypsin, unlike trypsin, failed to induce the characteristic fluorescence changes in a P9 Ser →Cys PAI-1 variant labeled with a nitrobenzofuran fluorescent probe (NBD) which were shown previously to report the serpin conformational change associated with active enzyme binding. These results demonstrate that a covalent interaction involving the proteinase catalytic serine contributes a major fraction of the binding energy to serpin-trypsin interactions and is essential for inducing the serpin conformational change involved in the trapping of enzyme in stable complexes. The contribution of a covalent bond to the stability of complexes of serine proteinases with inhibitors of the serpin family was evaluated by comparing the affinities of β-trypsin and the catalytic serine-modified derivative, β-anhydrotrypsin, for several serpin and non-serpin (Kunitz) inhibitors. Kinetic analyses showed that anhydrotrypsin had little or no ability to compete with trypsin for binding to α1-proteinase inhibitor (α1PI), plasminogen activator inhibitor 1 (PAI-1), antithrombin (AT), or AT-heparin complex when present at up to a 100-fold molar excess over trypsin. By contrast, equimolar levels of anhydrotrypsin blocked trypsin binding to non-serpin inhibitors. Equilibrium binding studies of inhibitor-enzyme interactions monitored by inhibitor displacement of the fluorescence probe, p-aminobenzamidine, from the enzyme active site, confirmed that the binding of serpins to anhydrotrypsin was undetectable in the case of α1PI or AT (KI > 10−5M), of low affinity in the case of AT-heparin complex (KI 7-9 × 10−6M), and of moderate affinity in the case of PAI-1 (KI 2 × 10−7M). This contrasted with the stoichiometric high affinity binding of the serpins to trypsin as well as of the non-serpin inhibitors to both trypsin and anhydrotrypsin. Maximal KI values for serpin-trypsin interactions of 1 to 8 × 10−11M, obtained from kinetic analyses of association and dissociation rate constants, indicated that the affinity of serpins for trypsin was minimally 4 to 6 orders of magnitude greater than that of anhydrotrypsin. Anhydrotrypsin, unlike trypsin, failed to induce the characteristic fluorescence changes in a P9 Ser →Cys PAI-1 variant labeled with a nitrobenzofuran fluorescent probe (NBD) which were shown previously to report the serpin conformational change associated with active enzyme binding. These results demonstrate that a covalent interaction involving the proteinase catalytic serine contributes a major fraction of the binding energy to serpin-trypsin interactions and is essential for inducing the serpin conformational change involved in the trapping of enzyme in stable complexes.