Rapid-reaction kinetic characterization of the pathway of streptokinase-plasmin catalytic complex formation

Abstract

Binding of the fibrinolytic proteinase plasmin (Pm) to streptokinase (SK) in a tight stoichiometric complex transforms Pm into a potent proteolytic activator of plasminogen. SK binding to the catalytic domain of Pm, with a dissociation constant of 12 pm, is assisted by SK Lys(414) binding to a Pm kringle, which accounts for a 11-20-fold affinity decrease when Pm lysine binding sites are blocked by 6-aminohexanoic acid (6-AHA) or benzamidine. The pathway of SK.Pm catalytic complex formation was characterized by stopped-flow kinetics of SK and the Lys(414) deletion mutant (SKDeltaK414) binding to Pm labeled at the active site with 5-fluorescein ([5F]FFR-Pm) and the reverse reactions by competitive displacement of [5F]FFR-Pm with active site-blocked Pm. The rate constants for the biexponential fluorescence quenching caused by SK and SKDeltaK414 binding to [5F]FFR-Pm were saturable as a function of SK concentration, reporting encounter complex affinities of 62-110 nm in the absence of lysine analogs and 4900-6500 and 1430-2200 nm in the presence of 6-AHA and benzamidine, respectively. The encounter complex with SKDeltaK414 was approximately 10-fold weaker in the absence of lysine analogs but indistinguishable from that of native SK in the presence of 6-AHA and benzamidine. The studies delineate for the first time the sequence of molecular events in the formation of the SK.Pm catalytic complex and its regulation by kringle ligands. Analysis of the forward and reverse reactions supports a binding mechanism in which SK Lys(414) binding to a Pm kringle accompanies near-diffusion-limited encounter complex formation followed by two slower, tightening conformational changes.

FIGURE 1.

Representative stopped-flow fluorescence traces of SK binding to [5F]FFR-Pm. A, fractional fluorescence changes (ΔF/F_o) (○) after rapid mixing of [5F]FFR-Pm and SK versus time in the absence of lysine analog (1) and in the presence of 50 mm 6-AHA (2) or 50 mm benzamidine (3). Final SK concentrations were 100, 100, and 50 nm, and [5F]FFR-Pm concentrations were 5, 20, and 10 nm, respectively. B, semilogarithmic plot of the normalized fluorescence data (○). Solid lines represent the least-squares fits of double exponentials. Stopped-flow time-traces were acquired and analyzed as described under “Experimental Procedures.”

FIGURE 2.

Dependence of the kinetics of [5F]FFR-Pm binding on SK and SKΔK414 concentration. A, dependences of k_{obs 1} (•, SK; ○, SKΔK414) and k_{obs 2} (▴, SK; ▵, SKΔK414) for the first and second phases of 5-20 nm [5F]FFR-Pm binding on the total SK or SKΔK414 concentration ([SK]_o) in the absence of lysine analogs. B, dependences of k_{obs 1} (•, SK; ○, SKΔK414) and k_{obs 2} (▴, SK; ▵, SKΔK414) on the total SK or SKΔK414 concentration in the presence of 50 mm 6-AHA. C, dependences of k_{obs 1} (•, SK; ○, SKΔK414) and k_{obs 2} (▴, SK; ▵, SKΔK414) on the total SK concentration in the presence of 50 mm benzamidine. Solid lines represent the least-squares fits by Equation 2, with the parameters given in Table 1. Dashed lines represent the least-squares fits using numerical integration of the reactions in Equation 4, with the parameters in Table 1. Experiments were performed and analyzed as described under “Experimental Procedures”.

FIGURE 3.

Kinetics of competitive dissociation of SK and SKΔK414 from the [5F]FFR-Pm complex by FFR-Pm. Increases in fluorescence after the addition of excess FFR-Pm to pre-equilibrated mixtures of [5F]FFR-Pm and SK or SKΔK414 due to dissociation of the of the SK·[5F]FFR-Pm complex. A, time courses for dissociation of the SK·[5F]FFR-Pm complex in the absence of effector (•). B, time courses in the presence of 50 mm 6-AHA (○) or 50 mm benzamidine (□). C, dissociation of the of the SKΔK414·[5F]FFR-Pm complex in the absence of effector (○), the presence of 50 mm 6-AHA (▵), or 50 mm benzamidine (□). Final concentrations of [5F]FFR-Pm, SK or SKΔK414, and FFR-Pm were 10, 100, and 910 nm, respectively. Solid lines represent the least-squares fits by a single exponential, with the parameters given in Table 1, and dashed lines represent the fits using the parameters obtained from nonlinear least-squares analysis of the mechanism in Equation 4 by numerical integration. D, the apparent off-rate as a function of increasing FFR-Pm concentration ([FFR-Pm]_o). The solid line represents simulation of a hyperbola using a K_D value of 12 pm and a limiting rate equal to the averaged k_off value of 0.042 min^-1 for reactions in the absence (•) and presence (○) of 50 mm 6-AHA. Experiments were performed and analyzed as described under “Experimental Procedures.”

FIGURE 4.

Simultaneous fits of SK concentration and time dependences based on numerical integration. Stopped-flow time-traces of the fractional change in fluorescence (ΔF/F_o) for reactions of 10 nm [5F]FFR-Pm with 0.025, 0.030, 0.052, 0.075, 0.1, 0.2, 0.35, 0.65, 0.9, 2, 5, 7, 10, and 14 μm SK in the absence of lysine analog (A). 0.1, 0.25, 0.4, 0.9, 2, 3, 5, 7, 10, and 15 μm SK in the presence of 50 mm 6-AHA (B), and 0.5, 1, 5 and 10 μm SK in the presence of 50 mm benzamidine (C). Data were collected during 0.5-30 s, until the reactions were >99% complete, and the fluorescence was stable. Data shown represent 92-99% completion of the reactions. Solid lines represent the least-squares fits to complete time traces using the mechanism in Equation 4, with the parameters listed in Table 1. Experiments were performed, and data were analyzed as described under “Experimental Procedures” and “Results.”

FIGURE 5.

Simultaneous fits of SKΔK414 concentration and time dependences based on numerical integration. Stopped-flow time-traces of the fractional change in fluorescence (ΔF/F_o) for reactions of 10 nm [5F]FFR-Pm with 0.050, 0.1, 0.25, 0.5, 1, 2, 5, and 14 μm SKΔK414 in the absence of lysine analog (A), 0.1, 0.25, 0.5, 1, 2, 5, 7.5, 10, and 14 μm SKΔK414 in the presence of 50 mm 6-AHA (B), and 1.25, 4, 6, 8, 10 and 14 μm SKΔK414 in the presence of 50 mm benzamidine (C). Data were collected during 0.5-30 s until the reactions were >99% complete, and the fluorescence was stable. Data shown represent 85-99% completion of the reactions. Solid lines represent the least-squares fits to complete time traces using the mechanism in Equation 4, with the parameters listed in Table 1. Experiments were performed and data were analyzed as described under “Experimental Procedures” and “Results.”

FIGURE 6.

Competitive equilibrium binding of SK and SKΔK414 to [5F]FFR-Pm and FFR-Pm in the presence of benzamidine. A, the fractional change in fluorescence (ΔF/F_o) of 2.5 nm [5F]FFR-Pm in buffer containing 50 mm benzamidine plotted as a function of the total SK concentration ([SK]_o) in the absence (•) and presence of 5.15 (○) and 15 nm (•) FFR-Pm. B, the fractional change in fluorescence (ΔF/F_o) of 5 nm [5F]FFR-Pm in buffer containing 50 mm benzamidine plotted as a function of the total SKΔK414 concentration ([SKΔK414]_o) in the absence (•) and presence of 10 nm (○) FFR-Pm. The solid lines represent least-squares fits of the cubic equation for competitive binding, with the parameters listed under “Results” and Table 1. Fluorescence titrations were performed and analyzed as described under “Experimental Procedures.”