Transient kinetics of heparin-catalyzed protease inactivation by antithrombin III. The reaction step limiting heparin turnover in thrombin neutralization.
The intrinsic protein fluorescence quenching which accompanies the heparin-accelerated inhibition of thrombin (T) by antithrombin III (AT) was resolved into a heparin-independent component associated with formation of the product T-AT complex and a component associated with an AT conformational change linked to heparin dissociation. To determine whether dissociation of heparin from the product T-AT complex limits the rate at which heparin can turn over catalytically, the kinetics of protein fluorescence quenching during the reaction of thrombin with AT X heparin complex (AT X H) were investigated by stopped-flow fluorimetry under pseudo-first order conditions ([AT X H]o much greater than [T]o). Both fluorescence components were quenched in a single exponential reaction with a hyperbolic dependence of the first order rate constant (kobs) on [AT X H]o. An indistinguishable hyperbolic dependence of kobs on [AT X H]o was measured by displacement of p-aminobenzamidine from the T active site, with both signals extrapolating to a limiting rate constant of 5 s-1. These results indicate that heparin dissociation occurs concomitant with T-AT complex formation at the limiting 5 s-1 rate constant. In reasonable agreement with this value, a kcat of 2.3 s-1 was determined for heparin turnover at catalytic concentrations. We conclude that formation of the T-AT complex is the primary rate-limiting step in heparin catalytic turnover and that this reaction is accompanied by a change in conformation of the AT component resulting in facile heparin dissociation.
D000818:Animals; D000990:Antithrombin III / Q000494:pharmacology*; D001550:Benzamidines / Q000494:pharmacology; D005470:Fluorometry; D006493:Heparin / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D008433:Mathematics; D011480:Protease Inhibitors / Q000494:pharmacology*; D013050:Spectrometry, Fluorescence; D013552:Swine; D013917:Thrombin / Q000037:antagonists & inhibitors*
D001550:Benzamidines; D011480:Protease Inhibitors; D000990:Antithrombin III; D006493:Heparin; D013917:Thrombin; C032157:benzamidine