ROLE OF THE CATALYTIC SERINE IN THE INTERACTIONS OF SERINE PROTEINASES WITH PROTEIN INHIBITORS OF THE SERPIN FAMILY - CONTRIBUTION OF A COVALENT INTERACTION TO THE BINDING-ENERGY OF SERPIN-PROTEINASE COMPLEXES

The contribution of a covalent bond to the stability of complexes of s erine proteinases with inhibitors of the serpin family was evaluated b y comparing the affinities of beta-trypsin and the catalytic serine-mo dified derivative, beta-anhydrotrypsin, for several serpin and non-ser pin (Kunitz) inhibitors. Kinetic analyses showed that anhydrotrypsin h ad little or no ability to compete with trypsin for binding to alpha(1 )-proteinase inhibitor (alpha(1)PI), 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. E quilibrium binding studies of inhibitor-enzyme interactions monitored by inhibitor displacement of the fluorescence probe, p-aminobenzamidin e, from the enzyme active site, confirmed that the binding of serpins to anhydrotrypsin was undetectable in the case of alpha(1)PI or AT (K- I > 10(-5) w), of low affinity in the case of AT-heparin complex (K(I) 7-9 x 10(-6) M), and of moderate affinity in the case of PAI-1 (K-I x 10(-7) M), This contrasted with the stoichiometric high affinity bindi ng of the serpins to trypsin as well as of the non-serpin inhibitors t o both trypsin and anhydrotrypsin, Maximal K-I values for serpin-tryps in interactions of 1 to 8 x 10(-11) M, obtained from kinetic analyses of association and dissociation rate constants, indicated that the aff inity gf serpins for trypsin was minimally 4 to 6 orders of magnitude greater than that of anhydrotrypsin. Anhydrotrypsin, unlike trypsin, f ailed 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 c hange 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 c hange involved in the trapping of enzyme in stable complexes.