Streptokinase binds to human plasmin with high affinity, perturbs the plasmin active site, and induces expression of a substrate recognition exosite for plasminogen.

Binding of streptokinase (SK) to plasminogen (Pg) conformationally activates the zymogen and converts both Pg and plasmin (Pm) into specific Pg activators. The interaction of SK with Pm and its relationship to the mechanism of Pg activation were evaluated in equilibrium binding studies with active site-labeled fluorescent Pm derivatives and in kinetic studies of SK-induced changes in the catalytic specificity of Pm. SK bound to fluorescein-labeled and native Pm with dissociation constants of 11 +/- 2 pm and 12 +/- 4 pm, which represented a 1,000-10,000-fold higher affinity than determined for Pg. Stoichiometric binding of SK to native Pm was followed by generation of a two-fragment form of SK cleaved at Lys(59) (SK'), which exhibited an indistinguishable affinity for labeled Pm, while a truncated, SK(55-414) species had a 120-360-fold reduced affinity. Binding of SK to native Pm was accompanied by a >50-fold enhancement in specificity for activation of Pg, which was paralleled by a surprising 2.6-10-fold loss of specificity of Pm for 8 of 11 tripeptide-pNA substrates. Further studies with Pm labeled at the active site with 2-anilinonaphthalene-6-sulfonic acid demonstrated directly that binding of SK to Pm resulted in expression of a new substrate binding exosite for Pg on the SK.Pm complex. It is concluded that SK activates Pg in part by preferential binding to the active zymogen conformation. High affinity binding of SK to Pm enhances Pg substrate specificity principally through emergence of a substrate recognition exosite.


Binding of streptokinase (SK) to plasminogen (Pg) conformationally activates the zymogen and converts both Pg and plasmin (Pm) into specific Pg activators.
The interaction of SK with Pm and its relationship to the mechanism of Pg activation were evaluated in equilibrium binding studies with active site-labeled fluorescent Pm derivatives and in kinetic studies of SK-induced changes in the catalytic specificity of Pm. SK bound to fluorescein-labeled and native Pm with dissociation constants of 11 ؎ 2 pM and 12 ؎ 4 pM, which represented a 1,000 -10,000-fold higher affinity than determined for Pg. Stoichiometric binding of SK to native Pm was followed by generation of a two-fragment form of SK cleaved at Lys 59 (SK), which exhibited an indistinguishable affinity for labeled Pm, while a truncated, SK 55-414 species had a 120 -360-fold reduced affinity. Binding of SK to native Pm was accompanied by a >50fold enhancement in specificity for activation of Pg, which was paralleled by a surprising 2.6 -10-fold loss of specificity of Pm for 8 of 11 tripeptide-pNA substrates. Further studies with Pm labeled at the active site with 2-anilinonaphthalene-6-sulfonic acid demonstrated directly that binding of SK to Pm resulted in expression of a new substrate binding exosite for Pg on the SK⅐Pm complex. It is concluded that SK activates Pg in part by preferential binding to the active zymogen conformation. High affinity binding of SK to Pm enhances Pg substrate specificity principally through emergence of a substrate recognition exosite.
Streptokinase (SK), 1 a 47,000 molecular weight protein from Streptococcus equisimilis, is used as a thrombolytic drug to activate plasminogen (Pg) into plasmin (Pm), the serine proteinase responsible for dissolution of fibrin clots (1,2). SK possesses no intrinsic enzyme activity, but binds specifically to Pg and Pm, converting both the zymogen and proteinase into Pg activators. SK binding to Pg results in the conformational expression of an active catalytic site on the zymogen that cleaves specifically the Arg 561 -Val 562 activation bond in the catalytic domain of Pg to form Pm (3)(4)(5)(6)(7). SK also binds to Pm, transforming the substrate specificity of the proteinase from one which is incapable of Pg activation into a specific activator (7)(8)(9), and decreasing greatly the reactivity of Pm toward its physiological serpin inhibitor, ␣ 2 -antiplasmin (10). The mechanism of conformational activation of Pg and the origin of the dramatic macromolecular substrate specificity change that Pm exhibits upon SK binding are unknown. The x-ray crystal structure of SK bound to the catalytic domain of Pm shows that SK consists of three, similarly-folded globular domains (11). SK surrounds the catalytic site, the three domains forming a "three-sided crater" with the active site of Pm at the bottom (11). The structure suggests that SK may induce a conformational change affecting the specificity of the catalytic site and/or participate directly in the binding of Pg as a substrate (11). However, the contribution of these two mechanisms to the change in Pm specificity has not been established. Structurefunction correlation studies with recombinant or proteolytic SK derivatives support a direct role for SK in recognition of Pg as a substrate (12)(13)(14). SK has been reported to cause modest changes in the kinetic constants for Pm with two peptide chromogenic substrates, suggesting that SK binding may also cause a change in catalytic specificity (9,15,16).
Quantitative equilibrium binding studies of SK interactions with Pg and Pm are required to define the mechanism of Pg activation and the origin of the SK-induced change in Pm substrate specificity. These studies are complicated by the coupling between the binding interactions and formation of Pm, and the ensuing additional proteolytic reactions catalyzed by Pm. Pm cleaves a 77-residue peptide from the amino terminus of native [Glu]Pg to form [Lys]Pg, which is accompanied by a conformational transition from a compact to an extended conformation that is activated to Pm more rapidly (2,17,18), and binds SK with enhanced affinity (19). SK also undergoes proteolysis by Pm, with early cleavage at Lys 59 generating modified SK (SKЈ), which consists of the noncovalent complex between the SK 1-59 and SK 60 -414 fragments, or similarly related fragments (13, 20 -23). Although the initial products retain activity in Pg activation, proteolysis at a number of other sites results ultimately in inactivation of SK. Intrinsic differences in the affinity of SK for Pg and Pm species accompanying these Pm-catalyzed reactions may contribute to the wide variation in estimates of the affinities of SK-Pg interactions from equilibrium binding studies. Binding studies using primarily surfaceimmobilized proteins have reported widely disparate dissociation constants for SK binding to [Glu]Pg ranging over ϳ10,000fold, from 28 to 220,000 pM. A similarly undefined range of values have been reported for Pm, and these studies found equivalent affinity of SK for Pg and Pm (10, 13, 19, 24 -27).
Fluorescent Pg analogs that are inactivated and covalently labeled at the zymogen catalytic site with probes were developed in previous studies to allow characterization of equilibrium binding interactions of SK in solution and without proteolysis occurring (19). In the present studies, this approach was extended to quantitatively characterize the interaction of SK with Pm under such conditions for the first time. Analysis of the fluorescence responses of an array of 16 active site-labeled fluorescent Pm derivatives shows that SK binds with an affinity that is 1,000 -10,000 higher than that determined previously for Pg (19). This finding supports the novel conclusion that SK conformationally activates Pg in part by stabilization of the active conformation of the zymogen. Stoichiometric binding of SK to native Pm is followed by appearance of SKЈ due to enhanced susceptibility of bound SK to cleavage by Pm. Comparison of the affinities of Pm for native SK, SKЈ, and a recombinant truncation mutant of SK reveals that cleavage at Lys 59 alone has little effect on Pm binding, whereas deletion of the amino-terminal 54 residues decreases the affinity greatly. Kinetic studies revealed that the emergence of macromolecular substrate specificity for Pg activation which accompanied binding of SK to Pm was surprisingly linked to an overall decrease in specificity for tripeptide-p-nitroanilide (pNA) substrates. Binding of Pg as a substrate of the SK⅐Pm complex was directly demonstrated to be due to expression of a substrate recognition exosite on the SK⅐Pm complex. The results support the conclusion that SK binding to Pm induces changes in the catalytic site affecting the substrate-binding subsites, but that these changes are not responsible for the enhanced specificity for activation of Pg, and that an exosite expressed on the SK⅐Pm complex mediates SK-induced recognition of Pg as a specific substrate.
Cleaved SK (SKЈ) was generated by incubation of 30 M Pg with equimolar SK in 50 mM Hepes, 0.125 M NaCl, 1 mM EDTA, 1 mg/ml PEG, pH 7.4, at 25°C for 10 min. One-tenth volume of 1 M Hepes, 10 mM 6-AHA, pH 7.0, was added, and Pm was inactivated by incubation with 200 M ATA-FFR-CH 2 Cl for 15 min. After dialysis against 50 mM Hepes, 0.3 M NaCl, 15 mM 6-AHA, 1 mM EDTA, 1 mg/ml PEG, pH 7.0, the inactivated SKЈ⅐Pm complex was immobilized onto iodoacetyl-(3,3Јiminobispropylamine)-agarose through the thiol group of the inhibitor (29). Immobilization was initiated by addition of 0.1 M NH 2 OH to the inactivated SKЈ⅐Pm complex in the presence of 8 ml of iodoacetyl-(3,3Јiminobispropylamine)-agarose, and followed by rapid mixing at room temperature for 2 h. The gel was washed with the above pH 7 buffer and SKЈ was eluted with buffer containing 3 M NaSCN. SKЈ was concen-trated with a YM-3 membrane and dialyzed against the buffer used for the experiments.
Wild-type recombinant SK and a mutant SK species lacking the amino-terminal 54 residues (SK 55-414 ) were those described by Fay and Bokka (30). The proteins were expressed in Escherichia coli BL21(DE3)pLysS and induced in log phase with 3 mM isopropyl-␤-Dthiogalactopyranoside for 4 h. The collected cells were suspended in 20 ml of pH 7.4 buffer lacking PEG, and containing phenylmethylsulfonyl fluoride (100 M), FFR-CH 2 Cl (1 M), FPR-CH 2 Cl (1 M), and aprotinin (1.6 g/ml), and lysed by three cycles of freezing and thawing, with addition of DNase I (200 g/ml) and MgCl 2 (80 mM) after the first and last cycles. SK species were precipitated from the lysate by addition of ammonium sulfate to 50% saturation and mixing at 4°C for 30 min. The precipitate was collected by centrifugation, dissolved in buffer containing the above inhibitors, and the precipitation was repeated twice. SK species were purified by affinity chromatography.
Gel Electrophoresis-SDS-polyacrylamide gradient gels (Bio-Rad) were stained with GELCODE Blue Stain (Pierce) or SYPRO Orange fluorescent protein stain (Molecular Probes). For studies of SK cleavage by Pm, reactions were initiated at 25°C by addition of Pm and stopped at various times by addition of 30 M FFR-CH 2 Cl before preparation of the gel samples.
Preparation of Active Site-labeled Pm-ATA-FFR-CH 2 Cl and ATA-FPR-CH 2 Cl were prepared as described previously (36 -38). Incorporation of fluorescein and AANS were quantitated using the previously determined coefficients (19,37). Stoichiometries of probe incorporation were 0.7-1.5 mol of probe/mol of Pm active sites for all of the derivatives. The derivatives were homogeneous by SDS-gel electrophoresis, with the exception of faint bands corresponding to unlabeled, disulfide-bonded dimeric products (38). Fluorescence Studies-Fluorescence measurements were made with an SLM 8100 spectrofluorometer in the ratio mode, using acrylic cuvettes coated with polyethylene glycol 20,000. Fluorescence titrations were measured at the excitation and emission wavelength difference maxima for the Pm derivatives, which were determined from spectra (4 nm band pass) in the absence and presence of 5 M SK. The excitation and emission wavelengths (nm) used in titrations (8 or 16 nm band pass) were: AANS, 332, 456; acrylodan, 396, 502; badan, 399, 502; BODIPY, 513, 525; 5-F and 6-F, 500, 516; OG, 492, 517; and RhX, 578, 601. These values were identical for the ATA-FFR-CH 2 Cl and ATA-FPR-CH 2 Cl derivatives, except for [AANS]FPR-Pm (332 nm excitation, 442 nm emission), and [RhX]FPR-Pm (578 nm excitation, 598 nm emission). Fluorescence titrations were performed by sequential addition of small volumes of SK to labeled Pm in 50 mM Hepes, 0.125 M NaCl, 1 mM EDTA, 1 mg/ml PEG, 1 mg/ml bovine serum albumin, 1 M FFR-CH 2 Cl, pH 7.4, at 25°C. Fluorescence changes were measured after equilibration for 5-10 min, and the measurements were expressed as the fractional change in the initial fluorescence ((F obs ϪF o )/F o ϭ ⌬F/F o ). Fluorescence changes were corrected for background by subtraction of measurements made on blanks lacking the labeled protein.
For titrations in which labeled Pm derivatives were screened for SKinduced fluorescence changes, concentrations of labeled Pm were 50 pM to 82 nM, which were the lowest concentrations that could be used for each label to give background signals of Ͻ20%. Titrations were analyzed by nonlinear least-squares fitting of the quadratic binding equation, with the maximum fluorescence change (⌬F max /F o ), dissociation constant (K D ), and stoichiometric factor (n) as the fitted parameters.
Measurements of the effect of native Pm on SK binding to [5-F]FFR-Pm were made following single additions of SK to cuvettes containing 75 pM [5-F]FFR-Pm and constant levels of native Pm, after a fixed incubation time of 10 min. The titrations were fit simultaneously by the equation for competitive binding of SK to native and labeled Pm to obtain the two dissociation constants, the stoichiometric factor for native Pm binding, and the maximum fluorescence change (40,41).
Chromogenic Substrate Kinetics-The effect of SK on the kinetic parameters for hydrolysis of tripeptide-pNA substrates by Pm was measured from the initial rates (Ͻ5% substrate depletion) at 405 nm and 25°C in 50 mM Hepes, 0.125 M NaCl, 1 mM EDTA, 1 mg/ml PEG, pH 7.4, in polyethylene glycol 20,000-coated cuvettes. All of the substrates were obtained from commercial sources except PGR-pNA which was synthesized by SynPep (Dublin, CA) and was 98% pure. Concentrations of peptide-pNA substrates were determined from the absorbance at 342 nm using an absorption coefficient of 8,266 M Ϫ1 cm Ϫ1 , and product concentrations were calculated using an absorption coefficient of 9,933 M Ϫ1 cm Ϫ1 (35,42). Reactions were initiated by addition of 0.25-100 nM Pm to buffer containing substrate in the absence or presence of a saturating concentration of SK (100 -500 nM). The results were fit by the Michaelis-Menten equation to obtain K m and k cat . Substrate concentrations ranged up to 2 mM, representing 0.3-19 ϫ K m .
The difference in Gibbs free energy between the transition state for a substrate containing a single amino acid substitution compared with that of a reference substrate was calculated as, where s and s ref are the specificity constants (k cat /K m ) for hydrolysis of the substrate and the reference substrate, respectively. ⌬⌬G subs was calculated by subtraction of ⌬G subs in the absence of SK from that in the presence of saturating SK. The Gibbs free energy for the SK-induced change in specificity for a single substrate (⌬G SK ) was calculated with Equation 2, where s and s SK are the specificity constants in the absence and presence of saturating SK, respectively. Initial rates of pyro-EPR-pNA hydrolysis as a function of Pm, SK, and substrate concentrations were analyzed with a model in which SK acts as a hyperbolic mixed-type modifier of Pm activity (Scheme 1; Ref. 43).
In this model, Pm and the SK⅐Pm complex bind substrate (S) with dissociation constants, K s and ␣K s , which are equal to K m and ␣K m , and generate product (P) with catalytic rate constants, k cat and ␤k cat . SK binds to Pm with dissociation constant, K D , and to the Pm-substrate complex (Pm⅐S) with dissociation constant, ␣K D .  (43), Because the assumption that [SK] f Х [SK] o was not valid in the case of a tight binding modifier under the conditions of the experiments, the results were analyzed by fitting of Equation 3, with the free SK concentrations calculated with Equations 4 and 5, where ⑀ is the absorption coefficient for pNA. This analysis gave the initial rate of chromogenic substrate hydrolysis at the beginning of the reaction (v 1 ) due to the Pm added initially, and the rate of the increase in activity with time due to bovine Pm formation (v 2 ). The dependence of v 2 on the total BPg concentration ([BPg] o ) and the concentration of the SK⅐Pm complex ([SK⅐Pm]) under conditions of low chromogenic substrate concentration was given by Equation 7, These first-order conditions for BPg activation were verified by the linear dependence of v 2 on BPg concentration up to 250 nM. Under these reaction conditions, the dependence of v 1 on SK concentration is given by the equations for the mixed modifier model described above, and the initial rate of the increase in activity due to BPm formation catalyzed by the SK⅐Pm complex is given by Equation 8, where k BPg /K BPg is the bimolecular rate constant for bovine Pg activation. The slow background rate of BPg activation by SK alone was Ͻ3% of the rate in the presence of Pm and was therefore neglected in the analysis. The initial velocities measured as a function of the total SK concentration at 0.1 nM Pm and 25 or 50 M chromogenic substrate were analyzed with Equations 3-5 and 8 to obtain the dissociation constant for SK binding to native Pm and the bimolecular rate constant for BPg activation by SK⅐Pm. The remaining kinetic constants were fixed at their determined values.
Least-squares fitting was performed with Scientist software (Micro-Math). Uncertainties in reported parameters are Ϯ2 S.D. Experimental error in the kinetic parameters was propagated.

RESULTS
Screening of Active Site-labeled Fluorescent Derivatives of Plasmin as Probes of Streptokinase Binding-To identify fluorescent derivatives of Pm for characterizing SK binding, an array of 16 derivatives was prepared by inactivation of Pm with ATA-FPR-CH 2 Cl or ATA-FFR-CH 2 Cl and labeling the thiol group generated on the amino terminus of the inhibitor with each of eight thiol-reactive probes (36,37). Characterization of SK Binding to [5-F]FFR-Pm-Analysis of the titrations in Fig. 1 indicated that SK bound with very high affinity, which precluded an accurate estimation of the dissociation constants for several of the derivatives labeled with less intensely fluorescent probes. Single titrations of the four fluorescein and one RhX derivatives, which could be done at low Pm concentrations (65-170 pM), yielded indistinguishable dissociation constants ranging from 5 Ϯ 7 pM to 15 Ϯ 9 pM (Fig. 1). The average stoichiometric factor determined from all of the titrations was 1.1 Ϯ 0.5 mol of SK/mol of Pm. On the basis of the high quantum yield of fluorescein and the large fluorescence change reported by [5-F]FFR-Pm on binding SK, this derivative was chosen for further studies of SK binding. Characterization of the Pm derivative confirmed that it was stoichiometrically labeled (1.1 Ϯ 0.1 mol of probe/mol of active sites) and that the probe was specifically incorporated into the catalytic site-containing light chain (Fig. 2, inset). Simultaneous fitting of SK titrations of [5-F]FFR-Pm at fixed concentrations of 75 pM, 1 nM, and 10 nM by the equation for a single binding interaction gave a dissociation constant of 11 Ϯ 2 pM, a stoichiometric factor of 1.3 Ϯ 0.1 mol of SK/mol of Pm, and a maximum fluorescence change of Ϫ48 Ϯ 1% (Fig. 2).
Proteolytic Cleavage of SK by Native Pm-To characterize the interaction of native Pm with SK, it was first necessary to evaluate proteolytic cleavage of SK by Pm. Incubation of 250 nM SK with an excess of Pm resulted in complete disappearance of native SK from 4 to 15% SDS gradient gels within 10 min and appearance of a new band with an 8,000 lower apparent molecular weight (Fig. 3). Results of similar experiments with 10 -20% gradient gels showed that the disappearance of native SK, which migrated with an apparent molecular weight of 49,000, was correlated with appearance of two new bands at apparent molecular weights of 44,000 and 6,000 (results not shown). The SK cleavage product was assigned to the noncovalent complex between SK 1-59 and SK 60 -414 (SKЈ) or a closely related secondary species on the basis of the apparent molecular weights and the results of previous studies identifying this and similar modified forms of SK (20 -22).
Reactions of a fixed concentration of SK with increasing concentrations of Pm in the 0.1-1 M range showed quantitative conversion of SK to SKЈ at equimolar levels of Pm and SK, and in the presence of up to a 4-fold excess of Pm (Fig. 3). When SK was present in excess, however, additional proteolysis products were observed (Fig. 3, A and B) that appeared to be the result of slower cleavage of free SK by the SK⅐Pm complex, on the basis that these were the predominant species present. To investigate this further, the effect of active site-blocked Pm (FFR-Pm) on cleavage of SK was examined. In reactions containing a 4-fold excess of SK over active Pm but no FFR-Pm, formation of SKЈ and the additional incomplete cleavage products were observed as before (Fig. 3C). When sufficient FFR-Pm was present to bind essentially all of the SK, the heterogeneous products were not observed and all of the SK was converted to SKЈ within 2 min (Fig. 3C). These results indicated that SK bound to Pm or FFR-Pm was preferentially cleaved at an enhanced rate by free Pm and the SK⅐Pm complex. Similar results were obtained at 20 -40 nM Pm and SK, the lowest concentrations that could be studied (results not shown).
Binding of SK to Native Pm-The above results indicated that SK complexes with native Pm were only transiently stable at Ն20 nM concentration. To characterize the binding of SK to native Pm and the possible effect of SK cleavage, [5-F]FFR-Pm was used as a probe to estimate the affinity of the initially formed complexes between native Pm and SK. The fluorescence of [5-F]FFR-Pm was stable (Ϯ3%) in the absence and presence of up to 1 nM native Pm for at least 40 min. Following addition of SK to mixtures of native and labeled Pm, the fluorescence decreased to a stable value within Յ5 min and increased gradually Յ10% over the following 20 -30 min. The steady state formed in the first 10 min was taken to represent equilibration of initially formed SK complexes with native and labeled Pm. Titrations of [5-F]FFR-Pm with SK in the absence and presence of 0.1, 0.2, and 1 nM native Pm (Fig. 4) were fit well by the equation for competitive binding of SK to native and labeled Pm, with a dissociation constant of 12 Ϯ 4 pM for SK binding to 1.3 Ϯ 0.3 sites on native Pm and an equivalent value of 16 Ϯ 4 pM for SK binding to [5-F]FFR-Pm (Fig. 4).
Binding of SKЈ and a Truncated SK Mutant to [5-F]FFR-Pm-To examine further the influence of SKЈ formation on its interaction with Pm, binding of SKЈ and a recombinant SK mutant lacking the amino-terminal 54 (SK  ) residues were examined. The SKЈ noncovalent complex was purified from a reaction mixture of Pg and SK, and contained both major fragments of SKЈ, along with smaller amounts of other degradation products evident by SDS-gel electrophoresis (Fig.  5, inset). SKЈ bound to labeled Pm with a dissociation constant of 43 Ϯ 1 pM and maximum fluorescence change of Ϫ44 Ϯ 1%, while wild-type recombinant SK bound with a dissociation constant of 33 Ϯ 6 pM and a 49 Ϯ 1% change (Fig. 5). Although these dissociation constants were 3-4-fold larger than the value of 12 Ϯ 4 pM obtained for native SK and Pm, they were considered indistinguishable because of the uncertainty inherent in accurate estimation of parameters for such high affinities, and the presence of some degradation products in the SKЈ preparation. By contrast, the purified SK truncation mutant, SK 55-414 bound [5-F]FFR-Pm with a dissociation constant of 4 Ϯ 1 nM, representing a loss of 120 -360 fold in affinity (Fig. 5). Moreover, SK 55-414 produced a 3-fold smaller fluorescence change of 16 Ϯ 1% compared with native SK, indicating a significant difference in the perturbations of the active site (Fig. 5). These results indicated that the cleaved, noncovalent SKЈ complex had nearly the same properties as native SK with respect to Pm interactions, whereas deletion of the 54 residues in SK 55-414 resulted in a large loss of affinity for Pm.
Effect of SK on the Specificity of Pm for Tripeptide Chromogenic Substrates-Michaelis-Menten kinetic parameters were determined for Pm and the SK⅐Pm complex for an array of 11 tripeptide-pNA substrates. For 8 of the substrates, significant decreases of 2.6 -10-fold in catalytic specificity (k cat /K m ) of Pm were observed upon SK binding, while 3 substrates were unaffected or decreased by less, and only CBO-RGR-pNA was increased, by 1.8-fold (Table I). The overall decrease in specificity was generally attributable to increases of 2-11-fold in K m , with smaller changes of ϳ2-fold in k cat . PGR-pNA, which has the sequence corresponding to the activation site in Pg cleaved specifically by SK⅐Pm was the poorest of the tripeptide substrates examined and the specificity of Pm for this substrate was reduced 2.6-fold by SK binding (Table I). The free energy differences calculated from the ratio of the specificity constants for Pm and SK⅐Pm (⌬G SK ) demonstrated that SK binding reduced the stability of the transition state by up to 1.4 kcal/mol, depending on the structure of the substrate (Table II).
The possible contribution of SKЈ formation to the observed changes in chromogenic substrate activity was investigated in extensive control experiments comparing the order of addition of the reactants and the effect of preincubation of SK and Pm on the rates. All of the reactions were linear over the 5-20-min duration of the assays, and the rates of pyro-EPR-pNA hydrolysis varied less than Ϯ7% for assays initiated with Pm or with substrate, over a range of Pm (0.1-1.0 nM), SK (0.1-100 nM), and substrate (50 -600 M) concentrations. The rates of reactions initiated with chromogenic substrate were similarly stable (Ϯ7%) over 20 -30 min of preincubation of SK and Pm. These results indicated that the changes in activity occurred rapidly on mixing SK and Pm and did not change significantly over the time course of the experiments. If cleavage of SK occurred during these experiments, it did not measurably affect the properties of the complexes with Pm.
To evaluate the relationship between SK binding and the specificity of Pm for the P1-P3 positions in the substrates, D-VLK-pNA, Tos-GPR-pNA, and PGR-pNA were chosen as reference substrates for comparison of free energy changes accompanying single residue substitutions (46,47). ⌬G subs was calculated for each substrate, which represented the difference in free energy of the transition states for the substrate and the reference substrate (Table II). The difference between ⌬G subs for Pm and SK⅐Pm (⌬⌬G subs ) represents the free energy for the SK-induced change in transition state stability relative to the reference substrate. Comparison of single substitutions of Arg for Lys at P1 in D-VLK-pNA and Lys for Arg in Tos-GPR-pNA showed little difference in the free energy change induced by SK, as given by ⌬⌬G subs , indicating little change in the preference of the S1 site as a result of SK binding (Table II). For Pm, favorable decreases in ⌬G subs were observed for a single substitution of Phe for Leu at P2 in D-VLK-pNA and for substitutions of both Phe at P2 and pyro-E at P3, whereas these effects were significantly less favorable for SK⅐Pm, demonstrating a differential effect of SK binding on the S2 subsite. Although substitutions at P3 resulted in significant changes in specificity, these were generally paralleled by Pm and SK⅐Pm, demonstrating little differential effect of SK binding (Table II). The above results were compatible with a mechanism in which SK acted as a tight-binding, mixed-type of modifier of Pm activity (see "Experimental Procedures"). The relationship between binding of SK to Pm and the change in substrate specificity was examined in more detailed kinetic studies with Tos-GPR-pNA and pyro-EPR-pNA. Titrations of 3 nM Pm with SK measured by the effect on Tos-GPR-pNA (100 M) hydrolysis indicated stoichiometric binding of SK, with a maximum decrease at saturating SK of 75 Ϯ 7% in the rate, in agreement with the 72% inhibition calculated from the independently determined kinetic parameters ( Table I). Comparison of the effects of native SK, SKЈ, and SK 55-414 on Pm with this substrate gave results that paralleled the properties of the SK species observed in the fluorescence studies (results not shown). Thus, native and wild-type recombinant SK decreased the initial rate maximally 75 and 73%, respectively, SKЈ produced a similar, 63% inhibition, whereas SK  produced no change at comparable concentrations, and ϳ38% inhibition only at 20-fold higher concentrations. The mixed-modifier mechanism was evaluated further by analysis of the effect of SK on hydrolysis of pyro-EPR-pNA, studied as a function of SK and substrate concentrations at 0.1 nM Pm (Fig. 6). The combined results were fit well by the mechanism with a dissociation constant of 11 Ϯ 5 pM for SK binding to native Pm, and kinetic constants that were indistinguishable from those determined in the previous experiments ( Fig. 6 and Table I). These results demonstrated the quantitative correspondence between high affinity SK binding to native Pm and its linkage with the decrease in specificity for peptide-pNA substrates.
Effect of SK Binding to Pm on Bovine Pg Activation-The relationship between SK binding to Pm and specificity for Pg activation was examined in kinetic studies of bovine Pg activation. Advantage was taken of the fact that bovine Pg is activated only very poorly by SK alone, but, like human Pg, is a specific substrate of the SK⅐Pm complex (7)(8)(9). For these experiments, the parabolic progress of pNA formation for reaction mixtures of Pm, SK, Tos-GPR-pNA, and BPg were analyzed to determine the initial rate of substrate hydrolysis at the beginning of the reaction due to the Pm present at zero time (v 1 ), and the rate of bovine plasmin formation (v 2 ) from the increase in activity with time. Low concentrations of chromogenic substrate and BPg were chosen to simplify the analysis (see "Experimental Procedures"). Under these conditions, the rate of Tos-GPR-pNA hydrolysis catalyzed by Pm (v 1 ) decreased to a non-zero limiting value in titrations with SK (Fig. 7A), as predicted from the inhibitory effect of SK as a mixed modifier of Pm activity with this substrate (Table I). The rate of BPg activation in the same reactions was undetectable in the absence of SK, and increased to a saturating value with SK concentration that represented an estimated Ͼ50-fold enhancement in the rate. Under the conditions used, the SK saturation curves in Fig. 7B represented the binding of SK to human Pm, independent of the chromogenic substrate and BPg concentrations (see "Experimental Procedures"). Analysis of the results in this manner gave a dissociation constant of 23 Ϯ 9 pM for SK binding and a bimolecular rate constant for activation of BPg at saturating SK of 8.4 Ϯ 0.3 ϫ 10 Ϫ5 nM Ϫ1 s Ϫ1 (Fig. 7B). These results demonstrated directly the correspondence between high affinity binding of SK to Pm, the loss of specificity for the chromogenic substrate, and the simultaneous expression of macromolecular substrate specificity for Pg.
Direct Evidence for Exosite-mediated Substrate Recognition of BPg by the SK⅐Pm Complex-Binding of BPg to [AANS]FPR-Pm was examined in the absence and presence of excess SK to evaluate the contribution of exosite interactions to BPg substrate recognition induced by SK binding to Pm. SK bound stoichiometrically to [AANS]FPR-Pm and enhanced the fluorescence maximally 84 Ϯ 1%. Subsequent titration with BPg produced a saturable further increase in the fluorescence of [AANS]FPR-Pm of 92 Ϯ 24% in the presence of saturating levels of SK (Fig. 8A). In the absence of SK, BPg produced no significant change in fluorescence (Ͻ3%, Fig. 8B) indicating no interaction of BPg with labeled Pm alone. Analysis of the results gave a dissociation constant of 2.9 Ϯ 1.3 M for BPg binding to the SK⅐[AANS]FPR-Pm complex. This value was independent of the presence of SK in excess of Pm (Fig. 8A) and indistinguishable from the K m determined under these conditions for BPg activation by the native SK⅐Pm complex of 3.6 Ϯ 1.3 M (results not shown). These results demonstrate that high affinity binding of SK to Pm is accompanied by expression of a substrate-binding exosite for BPg. This interaction must occur through an exosite because the S1-S3 specificity sites are blocked in [AANS]FPR-Pm by the fluorescent label. Exositemediated binding is principally responsible for BPg substrate recognition.  (19). Comparison of these results with the dissociation constant of 12 Ϯ 4 pM found here for Pm indicates that SK exhibits a dramatic, 1,000 -10,000-fold higher affinity for Pm compared with the Pg zymogen. The observation of indistinguishable dissociation constants for SK binding to four fluorescein and one rhodamine derivatives of Pm demonstrated that the affinity for SK was not significantly perturbed by the presence of the labels in the active site. Moreover, use of [5-F]FFR-Pm as a probe of competitive SK binding to native Pm yielded an indistinguishable dissociation constant. It should also be recognized, however, that there is disagreement among published studies concerning the dissociation constants for SK binding to Pg and Pm, which vary widely and some differ substantially from those reported here (10, 13, 24 -27). The dissociation constant of 50 pM inferred from the kinetic effect of SK binding on Pm inactivation by ␣ 2 -antiplasmin (10) is in agreement with the present direct binding studies. A potentially significant methodological difference that could contribute to the discrepancy in affinities is that surfaceimmobilized proteins were used primarily in other previous studies, whereas the present studies and that of Cederholm-Williams et al. (10) characterized the interactions in solution.
The SK-induced changes in fluorescence of the Pm derivatives may signal a conformational change affecting the microenvironment of the probes in the active site, and/or close proximity of SK to the probes in the complex. The variation of the probe responses to SK binding is thought to reflect changes in substrate specificity that affect the subsites occupied by the probe-tripeptide label, in combination with the individual re- The free energy differences in the stability of the transition states accompanying substitutions at P1-P3 in the reference substrates, D-VLK-pNA, Tos-GPR-pNA, and PGR-pNA are listed as ⌬G subs . Values were calculated from the specificity constants listed in Table I. The differences in free energy between Pm and SK ⅐ Pm for the substitutions in the reference substrates are listed as ⌬⌬G subs . The free energy for the SK-induced change in specificity for each substrate is listed as ⌬G SK . Substitutions in reference substrates are indicated in bold. Experiments were performed and thermodynamic parameters were calculated as described under "Experimental Procedures." porting properties of the probes. Consistent with this, the perturbation of the active site of Pm by SK was dependent on substitution of Phe for Pro in the P2 position of the linking inhibitor peptide for three probes, suggesting that the S2 subsite of Pm is affected by SK binding. Comparison of the results for SK and Pm with those of an analogous study of prothrombin fragment 2 binding to thrombin (37) shows a difference in the pattern of responses of the array of probes, which is thought to reflect the different substrate specificities of the enzymes and the structures of their active sites. In contrast to the results for SK and Pm, only 10 of 16 derivatives of thrombin exhibited a significant (Ͼ5%) change upon fragment 2 binding, and the effects of substitution of the P2 residue and probe were more dramatic, affecting both the sign and amplitude of the fluorescence changes (37). Similarly, binding of SK to Pm had a relatively consistent effect on peptide substrate specificity, inhibiting the enzyme for the majority of the substrates, while larger and more variable changes in kinetic parameters are associated with binding of fragment 2 and other ligands to thrombin (47,48). The behavior of thrombin reflects the restricted access of substrates to the deep catalytic site (49), and the associated dependence of thrombin substrate specificity on large, non-additive effects of neighboring S1-S3 subsite interactions (47,49). Plasmin is a much less specific proteinase than thrombin that shows little or no interdependence between specificity subsites (47). On this basis, the more uniform fluorescence and activity changes in the Pm catalytic site observed for SK binding are thought to reflect the initially less structured environment of the Pm active site, and possibly more homogeneous changes induced by SK. Analysis of the effect of SK on chromogenic substrate hydrolysis by Pm supported a mechanism in which SK acts as a tight binding mixed-modifier of Pm activity, binding to native Pm with the same dissociation constant as obtained in the fluorescence studies. Positive ⌬G SK values for 11 of 12 peptide substrates that were due mainly to increases in K m indicated that substrate binding to the active site opposes the change induced by SK for many substrates. This opposition may be steric because SK binds in the vicinity of the active site (11), but more likely represents a conformational change in Pm that reduces the affinity for substrate. Although this effect also predicts the likelihood of negative linkage between SK affinity and occupation of the active site by the peptide-probe fluorescent labels, this effect was evidently small for the Pm derivatives studied here. Examination of the differences in the stability of the transition states for pairs of Pm substrates accompanying the substitution of P1-P3 residues demonstrated the effects of SK on subsite specificity. Pm exhibits a slight preference for Lys over Arg at P1 (47), whereas the opposite specificity would presumably be optimal for cleavage of the Arg 561 -Val 562 bond required for Pg activation by the SK⅐Pm complex. The specificity of S1 for Arg or Lys, however, was not affected by SK. Substitutions at P2 had a differential effect on Pm and SK⅐Pm, corroborating the fluorescence results and indicating that the S2 specificity of free Pm and Pm bound to SK were uniquely different. Pm and the SK⅐Pm complex showed no significant differential changes in specificity for substitutions at P3.
Tight binding of SK to Pm and the resulting partial inhibition of chromogenic substrate activity were accompanied by a Ͼ50-fold enhanced appearance of specificity for activation of bovine Pg. The loss of specificity for chromogenic substrates included a tripeptide substrate with the Pro-Gly-Arg activation sequence of Pg, indicating that SK does not act by enhancing Pm specificity for Pg by redirecting it toward this particular sequence. Instead, it was concluded that changes in macromolecular specificity are facilitated by interactions more distant from S1-S3. Direct evidence supporting such an exosite-mediated mechanism of substrate recognition was obtained in the observation that BPg bound specifically to the SK complex formed with active site-labeled [AANS]FPR-Pm with an affinity that was the same as the K m for productive binding of BPg as a substrate of the native SK⅐Pm complex. The absence of interactions of BPg and [AANS]FPR-Pm alone and the independence of BPg binding on the presence of excess SK suggests that rearrangement of the flexible SK domains on binding to Pm may be required for expression of the BPg exosite. Exositemediated Pg binding is concluded to represent the dominant mechanism of the SK-induced change in macromolecular substrate specificity for Pg. Molecular modeling of Pg binding in a substrate mode to the SK⅐Pm complex supports this interpretation (11). Moreover, cofactor-mediated substrate binding is supported by the landmark studies of the structure of the functionally related ternary complex of staphylokinase with two molecules of plasmin bound. One of these is bound as the catalytic component and a second molecule of Pm is bound in the position Pg would occupy as the substrate of the staphylokinase-Pm complex (50). The many parallels between the structures of the staphylokinase-Pm and SK⅐Pm complexes and the functions of the cofactors in Pg activation support similar mechanisms of specific substrate recognition involving exositemediated cofactor interactions (50,51).
Stoichiometric binding of SK to native Pm was coupled with enhanced formation of SK cleaved at Lys 59 by Pm and SK⅐Pm, possibly due to a conformational change in SK which made this site more susceptible to cleavage. A number of approaches were taken to evaluate the influence of this proteolytic reaction on the interaction of SK with Pm. Purified preparations of SKЈ bound with similar affinity to [5-F]FFR-Pm, produced a fluorescence change of nearly the same amplitude as native SK, and had an effect similar to native SK on the chromogenic substrate activity of Pm. These results indicated little effect of the Lys 59 cleavage on the binding affinity, the change in active site environment, or substrate specificity changes. By contrast, a deletion mutant of SK in which 54 residues were removed from the amino terminus showed 120 -360-fold reduced affinity for Pm, raising the dissociation constant to a still significant value of 4 Ϯ 1 nM. This observation supports the conclusion that interactions of SK 1-59 and SK 60 -414 within the SKЈ complex and with Pm contribute substantially to the affinity of binding. Interestingly, interactions of the amino-terminal region of SK also play a role in perturbing the properties of the catalytic site, as evidenced by the large decrease in the amplitude of the fluorescence change for the SK  mutant. An incompletely resolved issue concerning SKЈ is the residual uncertainly about whether the results of the fluorescence and kinetic studies with native Pm reflect native SK, or SKЈ generated by Pm proteolysis. The mechanism of SKЈ formation appears to be through cleavage of SK bound to Pm by free Pm or by the SK⅐Pm complex, suggesting that at the low concentrations used in these experiments this process may have been slow, and the results may reflect the properties of native SK. While this is uncertain, the results of comparison of SK and SKЈ indicate that they have indistinguishable characteristics with respect to the properties studied here.
The large increase in affinity for SK accompanying the conversion of Pg to Pm provides new insight into the mechanism of conformational activation of Pg. The structure of SK bound to the catalytic domain of Pm shows that the SK ␥-domain makes critical contacts with part of the 142-152 segment of the chymotrypsinogen-homologous serine proteinase activation domain, in addition to interactions with other loops (11,52). The activation domain of chymotrypsinogen-like zymogens consists of the four segments 16 -19, 142-152, 184 -193, and 216 -223, which are disordered in the zymogen and undergo folding to form the active enzyme conformation (52). The activating conformational change is triggered by proteolytic generation of the new amino terminus at Ile 16 , and its insertion into the aminoterminal binding pocket (52,53). The energy cost of folding of the Pg sequence homologous to the 142-152 segment during the conversion of Pg to Pm may thus contribute to the higher affinity of SK for the active enzyme. Pg and other serine proteinase zymogens are thought to be in pre-existing equilibrium between inactive and active conformations (52,53) with equilibrium constants favoring the inactive conformation by 10 8 for trypsinogen (54) to about 12 for single chain tissue-type plasminogen activator (55). The Pg zymogen has ϳ10 5 -fold lower activity than Pm, indicating that the finding of a comparable, 10 3 -10 4 -fold increase in affinity of SK for Pm suggests that SK conformationally activates Pg in part by stabilizing the activated conformation of the zymogen. Further studies will be needed to determine whether the conformational change is induced by SK binding to Pg, and/or is the result of shifting the pre-existing unfavorable equilibrium toward the active zymogen conformation. The preferential affinity of SK for Pm also indicates that in the overall mechanism of activation of Pg by SK, the product of the reaction (Pm) binds to SK more tightly than Pg. It is speculated that this large increase in affinity plays an essential role in the catalytic mechanism by trapping the stable activated SK⅐Pm complex, which then recognizes Pg as a specific substrate through expression of the Pg-binding exosite, and proteolytically converts Pg to Pm.