The rate-determining step of the heparin-catalyzed antithrombin/thrombin reaction is independent of thrombin

The heparin-catalyzed inactivation of thrombin by antithrombin was examined using saturation kinetics and fractional reaction lifetimes. Based solely on kinetic analysis, the reaction binding sequence was determined to be heparin binding to antithrombin followed by binding of thrombin. Under readily accessible experimental conditions, the rate-determining step was zero order with respect to thrombin and varied from first order to zero order with respect to antithrombin as the concentration of antithrombin was increased. A saturation kinetics model was used to estimate a K(m) of 12 x 10^-8 M for formation of the kinetically active heparin-antithrombin complex, a k(cat) of 0.16 s^-1 for the rate-limiting step, and an overall rate constant of 13.3 x 10⁷ M^-1 ·s^-1 for heparin-antithrombin interaction.