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 serine proteinases with inhibitors of the serpin family was evaluated by comparing the affinities of beta-trypsin and the catalytic serine-modified derivative, beta-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 alpha 1-proteinase inhibitor (alpha 1PI), 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 alpha 1PI or AT (KI > 10(-5) M), of low affinity in the case of AT-heparin complex (KI 7-9 x 10(-6) M), and of moderate affinity in the case of PAI-1 (KI 2 x 10(-7) M). 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 x 10(-11) M, 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.
Center for Molecular Biology of Oral Diseases, University of Illinois, Chicago 60612-7213, USA.
S T
ST
Olson
P E
PE
Bock
J
J
Kvassman
J D
JD
Shore
D A
DA
Lawrence
D
D
Ginsburg
I
I
Björk
D000818:Animals; D000990:Antithrombin III / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D002417:Cattle; D006801:Humans; D007700:Kinetics; D008433:Mathematics; D008962:Models, Theoretical; D012694:Serine; D012697:Serine Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D015843:Serpins / Q000378:metabolism*; D014357:Trypsin / Q000737:chemistry / Q000378:metabolism*; D014361:Trypsin Inhibitors / Q000378:metabolism*
D015843:Serpins; D014361:Trypsin Inhibitors; C011661:anhydrotrypsin; D012694:Serine; D000990:Antithrombin III; D012697:Serine Endopeptidases; D014357:Trypsin