Domain interactions between streptokinase and human plasminogen.

Plasmin (Pm), the main fibrinolytic protease in the plasma, is derived from its zymogen plasminogen (Plg) by cleavage of a peptide bond at Arg(561)-Val(562). Streptokinase (SK), a widely used thrombolytic agent, is an efficient activator of human Plg. Both are multiple-domain proteins that form a tight 1:1 complex. The Plg moiety gains catalytic activity, without peptide bond cleavage, allowing the complex to activate other Plg molecules to Pm by conventional proteolysis. We report here studies on the interactions between individual domains of the two proteins and their roles in Plg activation. Individually, all three SK domains activated native Plg. While the SK alpha domain was the most active, its activity was uniquely dependent on the presence of Pm. The SK gamma domain also induced the formation of an active site in Plg(R561A), a mutant that resists proteolytic activation. The alpha and gamma domains together yielded synergistic activity, both in Plg activation and in Plg(R561A) active site formation. However, the synergistic activity of the latter was dependent on the correct N-terminal isoleucine in the alpha domain. Binding studies using surface plasmon resonance indicated that all three domains of SK interact with the Plg catalytic domain and that the beta domain additionally interacts with Plg kringle 5. These results suggest mechanistic steps in SK-mediated Plg activation. In the case of free Plg, complex formation is initiated by the rapid and obligatory interaction between the SK beta domain and Plg kringle 5. After binding of all SK domains to the catalytic domain of Plg, the SK alpha and gamma domains cooperatively induce the formation of an active site within the Plg moiety of the activator complex. Substrate Plg is then recognized by the activator complex through interactions predominately mediated by the SK alpha domain.     

Substrate kringle-mediated catalysis by the streptokinase-plasmin activator complex: Critical contribution of kringle-4 revealed by the mutagenesis approaches

Streptokinase (SK) is a protein co-factor with a potent capability for human plasminogen (HPG) activation. Our previous studies [1] have indicated a major role of long-range protein-protein contacts between the three domains (alpha, beta, and gamma) of SK and the multi-domain HPG substrate (K1-K5CD). To further explore this phenomenon, we prepared truncated derivatives of HPG with progressive removal of kringle domains, like K5CD, K4K5CD, K3-K5CD (K3K4K5CD), K2-K5CD (K2K3K4K5CD) and K1-K5CD (K1K2K3K4K5CD). While urokinase (uPA) cleaved the scissile peptide in the isolated catalytic domain (μPG) with nearly the same rate as with full-length HPG, SK-plasmin showed only 1-2% activity, revealing mutually distinct mechanisms of HPG catalysis between the eukaryotic and prokaryotic activators. Remarkably, with SK.HPN (plasmin), the ‘addition’ of both kringles 4 and 5 onto the catalytic domain showed catalytic rates comparable to full length HPG, thus identifying the dependency of the “long-range” enzyme-substrate interactions onto these two CD-proximal domains. Further, chimeric variants of K5CD were generated by swapping the kringle domains of HPG with those of uPA and TPA (tissue plasminogen activator), separately. Surprisingly, although native-like catalytic turnover rates were retained when either K1, K2 or K4 of HPG was substituted at the K5 position in K5CD, these were invariably lost once substituted with the evolutionarily more distant TPA- and uPA-derived kringles. The present results unveil a novel mechanism of SK.HPN action in which augmented catalysis occurs through enzyme-substrate interactions centered on regions in substrate HPG (kringles 4 and 5) that are spatially distant from the scissile peptide bond.     

Characterization of Lys-698-to-Met substitution in human plasminogen catalytic domain

Streptokinase (SK) is a human plasminogen (Pg) activator secreted by streptococci. The activation mechanism of SK differs from that of physiological Pg activators in that SK is not a protease and cannot proteolytically activate Pg. Instead, it forms a tight complex with Pg that proteolytically activates other Pg molecules. The residue Lys-698 of human Pg was hypothesized to participate in triggering activation in the SK-Pg complex. Here, we report a study of the Lys-698 to Met substitution in the catalytic domain of Pg (microPg) containing the proteolytic activation-resistant background (R561A). While it remains competent in forming a complex with SK, maintaining a comparable equilibration dissociation constant (K(D)), the recombinant protein shows a nearly 60-fold reduction in amidolytic activity relative to its R561A background when mixed with native SK. A 2.3 A crystal structure of this mutant microPg confirmed the correct folding of this recombinant protein. Combined with other biochemical data, these results support the premise that Lys-698 of human Pg plays a functional role in the so-called N-terminal insertion activation mechanism by SK.     

Effects on human plasminogen conformation and activation rate caused by interaction with VEK-30, a peptide derived from the group A streptococcal M-like protein (PAM)

In vertebrates, fibrinolysis is primarily carried out by the serine protease plasmin (Pm), which is derived from activation of the zymogen precursor, plasminogen (Pg). One of the most distinctive features of Pg/Pm is the presence of five homologous kringle (K) domains. These structural elements possess conserved Lys-binding sites (LBS) that facilitate interactions with substrates, activators, inhibitors and receptors. In human Pg (hPg), K2 displays weak Lys affinity, however the LBS of this domain has been implicated in an atypical interaction with the N-terminal region of a bacterial surface protein known as PAM (Pg-binding group A streptococcal M-like protein). A direct correlation has been established between invasiveness of group A streptococci and their ability to bind Pg. It has been previously demonstrated that a 30-residue internal peptide (VEK-30) from the N-terminal region of PAM competitively inhibits binding of the full-length parent protein to Pg. We have attempted to determine the effects of this ligand–protein interaction on the regulation of Pg zymogen activation and conformation. Our results show minimal effects on the sedimentation velocity coefficients (S°_20,w) of Pg when associated to VEK-30 and a direct relationship between the concentration of VEK-30 or PAM and the activation rate of Pg. These results are in contrast with the major conformational changes elicited by small-molecule activators of Pg, and point towards a novel mechanism of Pg activation that may underlie group A streptococcal (GAS) virulence.     

Regulation of nonproteolytic active site formation in plasminogen

Streptokinase may be less effective at saving lives in patients with heart attacks because it explosively generates plasmin in the bloodstream at sites distant from fibrin clots. We hypothesized that this rapid plasmin generation is due to SK's singular capacity to nonproteolytically generate the active protease SK x Pg*, and we examined whether the kringle domains regulate this process. An SK mutant lacking Ile-1 (deltaIle1-SK) does not form SK x Pg*, although it will form complexes with plasmin that can activate plasminogen. When compared to SK, deltaIle1-SK diminished the generation of plasmin in plasma by more than 30-fold, demonstrating that the formation of SK x Pg* plays an important role in SK activity in the blood. The rate of SK x Pg* formation (measured by an active site titrant) was much slower in Glu-Pg, which contains five kringle domains, than in Pg forms containing one kringle (mini-Pg) or no kringles (micro-Pg). In a similar manner, Streptococcus uberis Pg activator (SUPA), an SK-like molecule, generated SUPA x Pg* much slower with bovine Pg than bovine micro-Pg. The velocity of SK x Pg* formation was regulated by agents that influence the conformation of Pg through interactions with the kringle domains. Chloride ions, which maintain the compact Pg conformation, hindered SK x Pg* formation. In contrast, epsilon-aminocaproic acid, fibrin, and fibrinogen, which induce an extended Pg conformation, accelerated the formation of SK x Pg*. In summary, the explosive generation of plasmin in blood or plasma, which diminishes SK's therapeutic effects, is attributable to the formation of SK x Pg*, and this process is governed by kringle domains.     

Pro42 and Val45 of staphylokinase modulate intermolecular interactions of His43-Tyr44 pair and specificity of staphylokinase-plasmin activator complex

Staphylokinase (SAK) forms a 1:1 stoichiometric complex with plasmin (Pm) and changes its substrate specificity to create a plasminogen (Pg) activator complex. The His(43)-Tyr(44) pair of SAK resides within the active site cleft of the partner Pm and generates intermolecular contacts to confer Pg activator ability to the SAK-Pm bimolecular complex. Site-directed mutagenesis and molecular modeling studies unravelled that mutation at 42nd or 45th positions of SAK specifically disrupts cation-pi interaction of His(43) with Trp(215) of partner Pm within the active site, whereas pi-pi interaction of Tyr(44) with Trp(215) remain energetically favoured.     

Intermolecular interactions in staphylokinase-plasmin(ogen) bimolecular complex: function of His43 and Tyr44

Staphylokinase (SAK) forms a 1:1 stoichiometric complex with human plasmin (Pm) and switches its substrate specificity to generate a plasminogen (Pg) activator complex. Site-directed mutagenesis of SAKHis43 and SAKTyr44 demonstrated the crucial requirement of a positively charged and an aromatic residue, respectively, at these positions for optimal functioning of SAK-Pm activator complex. Molecular modeling studies further revealed the role of these residues in making cation-pi and pi-pi interactions with Trp215 of Pm and thus establishing the crucial intermolecular contacts within the active site cleft of the activator complex for the cofactor activity of SAK.     

alpha Domain deletion converts streptokinase into a fibrin-dependent plasminogen activator through mechanisms akin to staphylokinase and tissue plasminogen activator

The mechanism of action of plasminogen (Pg) activators may affect their therapeutic properties in humans. Streptokinase (SK) is a robust Pg activator in physiologic fluids in the absence of fibrin. Deletion of a "catalytic switch" (SK residues 1-59), alters the conformation of the SK alpha domain and converts SKDelta59 into a fibrin-dependent Pg activator through unknown mechanisms. We show that the SK alpha domain binds avidly to the Pg kringle domains that maintain Glu-Pg in a tightly folded conformation. By virtue of deletion of SK residues 1-59, SKDelta59 loses the ability to unfold Glu-Pg during complex formation and becomes incapable of nonproteolytic active site formation. In this manner, SKDelta59 behaves more like staphylokinase than like SK; it requires plasmin to form a functional activator complex, and in this complex SKDelta59 does not protect plasmin from inhibition by alpha(2)-antiplasmin. At the same time, SKDelta59 is unlike staphylokinase or SK and is more like tissue Pg activator, because it is a poor activator of the tightly folded form of Glu-Pg in physiologic solutions. SKDelta59 can only activate Glu-Pg when it was unfolded by fibrin interactions or by Cl(-)-deficient buffers. Taken together, these studies indicate that an intact alpha domain confers on SK the ability to nonproteolytically activate Glu-Pg, to unfold and process Glu-Pg substrate in physiologic solutions, and to alter the substrate-inhibitor interactions of plasmin in the activator complex. The loss of an intact alpha domain makes SKDelta59 activate Pg through classical "fibrin-dependent mechanisms" (akin to both staphylokinase and tissue Pg activator) that include: 1) a marked preference for a fibrin-bound or unfolded Glu-Pg substrate, 2) a requirement for plasmin in the activator complex, and 3) the creation of an activator complex with plasmin that is readily inhibited by alpha(2)-antiplasmin.     

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

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.     

Epsilon amino caproic acid inhibits streptokinase-plasminogen activator complex formation and substrate binding through kringle-dependent mechanisms

Lysine side chains induce conformational changes in plasminogen (Pg) that regulate the process of fibrinolysis or blood clot dissolution. A lysine side-chain mimic, epsilon amino caproic acid (EACA), enhances the activation of Pg by urinary-type and tissue-type Pg activators but inhibits Pg activation induced by streptokinase (SK). Our studies of the mechanism of this inhibition revealed that EACA (IC(50) 10 microM) also potently blocked amidolytic activity by SK and Pg at doses nearly 10000-fold lower than that required to inhibit the amidolytic activity of plasmin. Different Pg fragments were used to assess the role of the kringles in mediating the inhibitory effects of EACA: mini-Pg which lacks kringles 1-4 of Glu-Pg and micro-Pg which lacks all kringles and contains only the catalytic domain. SK bound with similar affinities to Glu-Pg (K(A) = 2.3 x 10(9) M(-1)) and to mini-Pg (K(A) = 3.8 x 10(9) M(-)(1)) but with significantly lower affinity to micro-Pg (K(A) = 6 x 10(7) M(-)(1)). EACA potently inhibited the binding of Glu-Pg to SK (K(i) = 5.7 microM), but was less potent (K(i) = 81.1 microM) for inhibiting the binding of mini-Pg to SK and had no significant inhibitory effects on the binding of micro-Pg and SK. In assays simulating substrate binding, EACA also potently inhibited the binding of Glu-Pg to the SK-Glu-Pg activator complex, but had negligible effects on micro-Pg binding. Taken together, these studies indicate that EACA inhibits Pg activation by blocking activator complex formation and substrate binding, through a kringle-dependent mechanism. Thus, in addition to interactions between SK and the protease domain, interactions between SK and the kringle domain(s) play a key role in Pg activation.     

Fibrin‐targeted plasminogen activation by plasminogen activator,PadA, from Streptococcus dysgalactiae

Bacterial plasminogen activators differ from each other in their mechanism of plasminogen activation besides their host specificity. Three‐domain streptokinase (SK) and two‐domain PauA generate nonproteolytic active site center in their cognate partner plasminogen but their binary activator complexes are resistant to α2‐antiplasmin (a2AP) inhibition causing nonspecific plasminogen activation in plasma. In contrast, single‐domain plasminogen activator, staphylokinase (SAK), requires proteolytic cleavage of human plasminogen into plasmin for the active site generation, and this activator complex is inhibited by a2AP. The single‐domain plasminogen activator, PadA, from Streptococcus dysgalatiae, having close sequence and possible structure homology with SAK, was recently reported to activate bovine Pg in a nonproteolytic manner similar to SK. We report hereby that the binary activator complex of PadA with bovine plasminogen is inhibited by a2AP and PadA is recycled from this complex to catalyze the activation of plasminogen in the clot environment, where it is completely protected from a2AP inhibition. Catalytic efficiency of the activator complex formed by PadA and bovine plasminogen is amplified several folds in the presence of cyanogen bromide digested fibrinogen but not by intact fibrinogen indicating that PadA may be highly efficient at the fibrin surface. The present study, thus, demonstrates that PadA is a unique single‐domain plasminogen activator that activates bovine plasminogen in a fibrin‐targeted manner like SAK. The sequence optimization by PadA for acquiring the characteristics of both SK and SAK may be exploited for the development of efficient and fibrin‐specific plasminogen activators for thrombolytic therapy.     

Effects of deletion of streptokinase residues 48-59 on plasminogen activation

Streptokinase (SK) is a thrombolytic agent widely used for the clinical treatment of clotting disorders such as heart attack. The treatment is based on the ability of SK to bind plasminogen (Pg) or plasmin (Pm), forming complexes that proteolytically activate other Pg molecules to Pm, which carries out fibrinolysis. SK contains three major domains. The N-terminal domain, SKalpha, provides the complex with substrate recognition towards Pg. SKalpha contains a unique mobile loop, residues 45-70, absent in the corresponding domains of other bacterial Pg activators. To study the roles of this loop, we deleted 12 residues in this loop in both full-length SK and the SKalpha fragment. Kinetic data indicate that this loop participates in the recognition of substrate Pg, but does not function in the active site formation in the activator complex. Two crystal structures of the deletion mutant of SKalpha (SKalpha(delta)) complexed with the protease domain of Pg were determined. While the structure of SKalpha(delta) is essentially the same as this domain in full-length SK, the mode of SK-Pg interaction was however different from a previously observed structure. Even though mutagenesis studies indicated that the current complex represents a minor interacting form in solution, the binding to SKalpha(delta) triggered similar conformational changes in the Pg active site in both crystal forms.     

Computational simulations assessment of mutations impact on streptokinase (SK) from a group G streptococci with enhanced activity – insights into the functional roles of structural dynamics flexibility of SK and stabilization of SK–μplasmin catalytic complex

Streptokinase (SK), a plasminogen activator (PA) that converts inactive plasminogen (Pg) to plasmin (Pm), is a protein secreted by groups A, C, and G streptococci (GAS, GCS, and GGS, respectively), with high sequence divergence and functional heterogeneity. While roles of some residual changes in altered SK functionality are shown, the underlying structural mechanisms are less known. Herein, using computational approaches, we analyzed the conformational basis for the increased activity of SK from a GGS (SKG132) isolate with four natural residual substitutions (Ile33Phe, Arg45Gln, Asn228Lys, Phe287Ile) compared to the standard GCS (SKC). Using the crystal structure of SK.Pm catalytic complex as main template SKC.μPm catalytic complex was modeled through homology modeling process and validated by several online validation servers. Subsequently, SKG132.μPm structure was constructed by altering the corresponding residual substitutions. Results of three independent MD simulations showed increased RMSF values for SKG132.μPm, indicating the enhanced structural flexibility compared to SKC.μPm, specially in 170 and 250 loops and three regions: R1 (149–161), R2 (182–215) and R3 (224–229). In parallel, the average number of Hydrogen bonds in 170 loop, R2 and R3 (especially for Asn228Lys) of SKG132 compared to that of the SKC was decreased. Accordingly, residue interaction networks (RINs) analyses indicated that Asn228Lys might induce more level of structural flexibility by generation of free Lys256, while Phe287Ile and Ile33Phe enhanced the stabilization of the SKG132.μPm catalytic complex. These results denoted the potential role of the optimal dynamic state and stabilized catalytic complex for increased PA potencies of SK as a thrombolytic drug.     

Substrate kringle-mediated catalysis by the streptokinase-plasmin activator complex: critical contribution of kringle-4 revealed by the mutagenesis approaches

Streptokinase (SK) is a protein co-factor with a potent capability for human plasminogen (HPG) activation. Our previous studies [1] have indicated a major role of long-range protein-protein contacts between the three domains (alpha, beta, and gamma) of SK and the multi-domain HPG substrate (K1-K5CD). To further explore this phenomenon, we prepared truncated derivatives of HPG with progressive removal of kringle domains, like K5CD, K4K5CD, K3-K5CD (K3K4K5CD), K2-K5CD (K2K3K4K5CD) and K1-K5CD (K1K2K3K4K5CD). While urokinase (uPA) cleaved the scissile peptide in the isolated catalytic domain (μPG) with nearly the same rate as with full-length HPG, SK-plasmin showed only 1-2% activity, revealing mutually distinct mechanisms of HPG catalysis between the eukaryotic and prokaryotic activators. Remarkably, with SK.HPN (plasmin), the 'addition' of both kringles 4 and 5 onto the catalytic domain showed catalytic rates comparable to full length HPG, thus identifying the dependency of the "long-range" enzyme-substrate interactions onto these two CD-proximal domains. Further, chimeric variants of K5CD were generated by swapping the kringle domains of HPG with those of uPA and TPA (tissue plasminogen activator), separately. Surprisingly, although native-like catalytic turnover rates were retained when either K1, K2 or K4 of HPG was substituted at the K5 position in K5CD, these were invariably lost once substituted with the evolutionarily more distant TPA- and uPA-derived kringles. The present results unveil a novel mechanism of SK.HPN action in which augmented catalysis occurs through enzyme-substrate interactions centered on regions in substrate HPG (kringles 4 and 5) that are spatially distant from the scissile peptide bond.     

Role of the N-terminal region of staphylokinase (SAK): evidence for the participation of the N-terminal region of SAK in the enzyme-substrate complex formation

Staphylokinase (SAK) forms an inactive 1:1 complex with plasminogen (PG), which requires both the conversion of PG to plasmin (Pm) to expose an active site in PG-SAK activator complex and the amino-terminal processing of SAK to expose the positively charged (Lys-11) amino-terminus after removal of the 10 N-terminal amino acid residues from the full length protein. The mechanism by which the N-terminal segment of SAK affects its PG activation capability was investigated by generating SAK mutants, blocked in the native amino-terminal processing site of SAK, and carrying an alteration in the placement of the positively charged amino acid residue, Lys-11, and further studying their interaction with PG, Pm, miniplasmin and kringle structures. A ternary complex formation between PG-SAK PG was observed when an immobilized PG-SAK binary complex interacted with free radiolabelled PG in a sandwich binding experiment. Formation of this ternary complex was inhibited by a lysine analog, 6-aminocaproic acid (EACA), in a concentration dependent manner, suggesting the involvement of lysine binding site(s) in this process. In contrast, EACA did not significantly affect the formation of binary complex formed by native SAK or its mutant derivatives. Furthermore, the binary (activator) complex formed between PG and SAK mutant, PRM3, lacking the N-terminal lysine 11, exhibited 3-4-fold reduced binding with PG, Pm or miniplasmin substrate during ternary complex formation as compared to native SAK. Additionally, activator complex formed with PRM3 failed to activate miniplasminogen and exhibited highly diminished activation of substrate PG. Protein binding studies indicated that it has 3-5-fold reduction in ternary complex formation with miniplasmin but not with the kringle structure. In aggregate, these observations provide experimental evidence for the participation of the N-terminal region of SAK in accession and processing of substrate by the SAK-Pm activator complex to potentiate the PG activation by enhancing and/or stabilizing the interaction of free PG.     

Functional roles of streptokinase C-terminal flexible peptide in active site formation and substrate recognition in plasminogen activation

The bacterial protein streptokinase (SK) activates human plasminogen (Pg) into the fibrinolytic protease plasmin (Pm). Roughly 40 residues from the SK C-terminal domain are mobile in the crystal structure of SK complexed with the catalytic domain of Pm, and the functions of this C-tail remain elusive. To better define its roles in Pg activation, we constructed and characterized three C-terminal truncation mutants containing SK residues 1-378, 1-386, and 1-401, respectively. They exhibit gradually reduced amidolytic activity and Pg-activator activity, as well as marginally decreased binding affinity toward Pg, as more of the C-terminus is deleted. As compared with full-length SK, the shortest construct, SK(1-378), exhibits an 80% decrease in amidolytic activity (k(cat)/K(M)), an 80% decrease in Pg-activator activity, and a 30% increase in the dissociation constant toward the Pg catalytic domain. The C-terminal truncation mutations did not attenuate the resistance of the SK-Pm complex to alpha(2)-antiplasmin. Attempts at using a purified C-tail peptide to rescue the activity loss of the truncation mutants failed, suggesting that the integrity of the SK C-terminal peptide is important for the full function of SK.     

Degradation of streptokinase and the catalytic properties of the plasmin-streptokinase complex

Streptokinase (SK) interacts with human plasminogen (Pg) or plasmin (Pm) with formation of Pg-SK or Pm-SK complex. Pm-SK complex manifests a fibrinolytic, amidolytic and Pg activator activity. SK in complex with Pm isn't stable and so capable to be hydrolysed rapidly. We investigated a correlation between molecular form of SK and catalytic properties of equimolar Pm-SK complex during preincubation at 20 degrees C. It was found out that amidolytic activity of Pm-SK complex was not changing for 5 hours and decreased to the initial Pm value after 24 hours. During this time alpha 2-antiplasmin (alpha 2-AP) has any effect on amidolytic activity of the complex. Fibrinolytic activity of Pm-SK complex makes up 20% of the initial Pm value and wasn't changing within the investigated period. Pg activator activity was decreasing rapidly to 30-40% of the initial one within few minutes from the moment of Pm-SK complex formation. It was 10-20% of that initial after 24 hours. The decrease in Pg activator activity of Pm-SK complex correlated with the initial very rapid conversion of 47 kDa SK to 36 kDa SK within few minutes and following more slow conversion of SK in 31, 25 and 15 kDa fragments after 5 hours. alpha 2-AP didn't influence on the Pg activator activity of Pm-SK complex but eliminated its fibrinolytic activity completely. It was supposed that alpha 2-AP inhibited fibrinolytic activity of Pm-SK complex similarly to 6-aminohexanoic acid by preventing Pm-SK complex binding to fibrin polymer.     

Role of the streptokinase alpha-domain in the interactions of streptokinase with plasminogen and plasmin

The role of the streptokinase (SK) alpha-domain in plasminogen (Pg) and plasmin (Pm) interactions was investigated in quantitative binding studies employing active site fluorescein-labeled [Glu]Pg, [Lys]Pg, and [Lys]Pm, and the SK truncation mutants, SK-(55-414), SK-(70-414), and SK-(152-414). Lysine binding site (LBS)-dependent and -independent binding were resolved from the effects of the lysine analog, 6-aminohexanoic acid. The mutants bound indistinguishably, consistent with unfolding of the alpha-domain on deletion of SK-(1-54). The affinity of SK for [Glu]Pg was LBS-independent, and although [Lys]Pg affinity was enhanced 13-fold by LBS interactions, the LBS-independent free energy contributions were indistinguishable. alpha-Domain truncation reduced the affinity of SK for [Glu]Pg 2-7-fold and [Lys]Pg 相似文献   

Rapid Binding of Plasminogen to Streptokinase in a Catalytic Complex Reveals a Three-step Mechanism

Rapid kinetics demonstrate a three-step pathway of streptokinase (SK) binding to plasminogen (Pg), the zymogen of plasmin (Pm). Formation of a fluorescently silent encounter complex is followed by two conformational tightening steps reported by fluorescence quenches. Forward reactions were defined by time courses of biphasic quenching during complex formation between SK or its COOH-terminal Lys⁴¹⁴ deletion mutant (SKΔK414) and active site-labeled [Lys]Pg ([5-(acetamido)fluorescein]-d-Phe-Phe-Arg-[Lys]Pg ([5F]FFR-[Lys]Pg)) and by the SK dependences of the quench rates. Active site-blocked Pm rapidly displaced [5F]FFR-[Lys]Pg from the complex. The encounter and final SK·[5F]FFR-[Lys]Pg complexes were weakened similarly by SK Lys⁴¹⁴ deletion and blocking of lysine-binding sites (LBSs) on Pg kringles with 6-aminohexanoic acid or benzamidine. Forward and reverse rates for both tightening steps were unaffected by 6-aminohexanoic acid, whereas benzamidine released constraints on the first conformational tightening. This indicated that binding of SK Lys⁴¹⁴ to Pg kringle 4 plays a role in recognition of Pg by SK. The substantially lower affinity of the final SK·Pg complex compared with SK·Pm is characterized by a ∼25-fold weaker encounter complex and ∼40-fold faster off-rates for the second conformational step. The results suggest that effective Pg encounter requires SK Lys⁴¹⁴ engagement and significant non-LBS interactions with the protease domain, whereas Pm binding additionally requires contributions of other lysines. This difference may be responsible for the lower affinity of the SK·Pg complex and the expression of a weaker “pro”-exosite for binding of a second Pg in the substrate mode compared with SK·Pm.     

链激酶研究概况及其进展

链激酶(Streptok inase,SK)是世界上最早发现的纤维蛋白酶原激活剂,也是最早作为临床药品治疗血栓性疾病的溶栓酶。它是由A,C,G群链球菌中β-溶血性链球菌分泌的胞外非酶蛋白质,能和纤溶酶原结合,将纤溶酶原激活为纤溶酶,具有溶解血栓的作用。本文详细综述了该酶的性质、在溶栓酶中的地位、研究历史、作用机理等。此外,由于它有半衰期短、不具有纤维蛋白特异性、治疗后出血和血栓易复发的缺点,所以有必要用基因工程的手段进行改造,以达到更好的治疗效果。