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.
Olson
S T
ST
Center for Molecular Biology of Oral Diseases, University of Illinois, Chicago 60612-7213, USA.
Bock
P E
PE
Kvassman
J
J
Shore
J D
JD
Lawrence
D A
DA
Ginsburg
D
D
Björk
I
I
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