Binding of the COOH-terminal lysine residue of streptokinase to plasmin(ogen) kringles enhances formation of the streptokinase.plasmin(ogen) catalytic complexes

Abstract

Streptokinase (SK) activates human fibrinolysis by inducing non-proteolytic activation of the serine proteinase zymogen, plasminogen (Pg), in the SK.Pg* catalytic complex. SK.Pg* proteolytically activates Pg to plasmin (Pm). SK-induced Pg activation is enhanced by lysine-binding site (LBS) interactions with kringles on Pg and Pm, as evidenced by inhibition of the reactions by the lysine analogue, 6-aminohexanoic acid. Equilibrium binding analysis and [Lys]Pg activation kinetics with wild-type SK, carboxypeptidase B-treated SK, and a COOH-terminal Lys414 deletion mutant (SKDeltaK414) demonstrated a critical role for Lys414 in the enhancement of [Lys]Pg and [Lys]Pm binding and conformational [Lys]Pg activation. The LBS-independent affinity of SK for [Glu]Pg was unaffected by deletion of Lys414. By contrast, removal of SK Lys414 caused 19- and 14-fold decreases in SK affinity for [Lys]Pg and [Lys]Pm binding in the catalytic mode, respectively. In kinetic studies of the coupled conformational and proteolytic activation of [Lys]Pg, SKDeltaK414 exhibited a corresponding 17-fold affinity decrease for formation of the SKDeltaK414.[Lys]Pg* complex. SKDeltaK414 binding to [Lys]Pg and [Lys]Pm and conformational [Lys]Pg activation were LBS-independent, whereas [Lys]Pg substrate binding and proteolytic [Lys]Pm generation remained LBS-dependent. We conclude that binding of SK Lys414 to [Lys]Pg and [Lys]Pm kringles enhances SK.[Lys]Pg* and SK.[Lys]Pm catalytic complex formation. This interaction is distinct structurally and functionally from LBS-dependent Pg substrate recognition by these complexes.

FIGURE 1. Effect of removal of the COOH-terminal Lys⁴¹⁴ of SK on binding to [Glu]Pg, [Lys]Pg, and [Lys]Pm

A, effect of CpB treatment of native SK on affinity for [5F]FFR-[Lys]Pg in the absence and presence of 6-AHA. Titrations of the decrease in fluorescence (ΔF/F_o) of 15 nM [5F]FFR-[Lys]Pg as a function of total SK concentration ([SK]_o) for native SK in the absence (●) and presence (○) of 100 mM 6-AHA and CpB-treated SK in the absence (△) and presence (▲) of 6-AHA. B, titrations of 15 nM fluorescein-labeled [Lys]Pg with wtSK in the absence (●) and presence (○) of 10 mM 6-AHA and with SKΔK414 in the absence (□) and presence (■) of 10 mM 6-AHA. C and D, analogous titrations of 15 nM fluorescein-labeled [Glu]Pg (C) and 75 pM fluorescein-labeled [Lys]Pm (D) using the same symbols as in B. Lines represent the least squares fits with the parameters given in the “Results and Discussion.” Fluorescence titrations were performed and analyzed as described under “Experimental Procedures.”

FIGURE 2. Competitive binding of native [Lys]Pg and [5F]FFR-[Lys]Pg to wtSK and SKΔK414

A, the fractional change in fluorescence (ΔF/F_o) of 15 nM [5F]FFR-[Lys]Pg plotted against the total concentration of native [Lys]Pg ([Pg]_o) for 150 nM (●) and 1 μM (○) wtSK. B, similar titrations are shown in the presence of 10 mM 6-AHA for 100 nM (▲) and 1 μM (△) wtSK. C, titrations at 1 μM SKΔK414 in the absence (■) and presence (□) of 10 mM 6-AHA. The lines represent the fit by the cubic competitive binding equation with the parameters given in the “Results and Discussion” and n fixed at 1. Fluorescence titrations were performed and analyzed as described under “Experimental Procedures.”

FIGURE 3. Kinetics of [Lys]Pg activation by wtSK and SKΔK414

A, dependence of v₁ on the total SK species concentration ([SK]_o) obtained from reactions of 15 nM [Lys]Pg in the presence of 200 μM VLK-pNA and in the absence (●) and presence (○) of 10 mM 6-AHA and with SKΔK414 in the absence (■) and presence (□) of 10 mM 6-AHA. B, SK concentration dependencies of v₂ rates for wtSK in the absence (●) and presence (○) of 10 mM 6-AHA and SKΔK414 in the absence (■) and presence (□) of 10 mM 6-AHA. Lines represent the fits by the equations described previously (5, 6) with the parameters given in the “Results and Discussion.” Activation reactions were performed and analyzed as described under “Experimental Procedures.”

SCHEME 1