Deletion of Ile1 Changes the Mechanism of Streptokinase: Evidence for the Molecular Sexuality Hypothesis

The selective deletion of a discrete surface-exposed epitope (residues 254 -262; 250-loop) in the ␤ domain of streptokinase (SK) significantly decreased the rates of substrate human plasminogen (HPG) activation by the mutant (SK del254 -262 ). A kinetic analysis of SK del254 -262 revealed that its low HPG activator activity arose from a 5-6-fold increase in K m for HPG as substrate, with little alteration in k cat rates. This increase in the K m for the macromolecular substrate was proportional to a similar decrease in the binding affinity for substrate HPG as observed in a new resonant mirror-based assay for the real-time kinetic analysis of the docking of substrate HPG onto preformed binary complex. In contrast, studies on the interaction of the two proteins with microplasminogen showed no difference between the rates of activation of microplasminogen under conditions where HPG was activated differentially by nSK and SK del254 -262 . The involvement of kringles was further indicated by a hypersusceptibility of the SK del254 -262 ⅐ plasmin activator complex to ⑀-aminocaproic acid-mediated inhibition of substrate HPG activation in comparison with that of the nSK ⅐ plasmin activator complex. Further, ternary binding experiments on the resonant mirror showed that the binding affinity of kringles 1-5 of HPG to SK del254 -262 ⅐ HPG was reduced by about 3-fold in comparison with that of nSK⅐HPG. Overall, these observations identify the 250 loop in the ␤ domain of SK as an important structural determinant of the inordinately stringent substrate specificity of the SK⅐HPG activator complex and demonstrate that it promotes the binding of substrate HPG to the activator via the kringle(s) during the HPG activation process. Streptokinase (SK),1 a bacterial protein secreted by the Lancefield Group C ␤-hemolytic streptococci, is widely used as a thrombolytic agent in the treatment of various circulatory disorders, including myocardial infarction (1). Unlike other human plasminogen (HPG) activators, like tissue plasminogen activator and urokinase, SK does not possess any intrinsic enzymatic activity. Instead, SK forms an equimolar, stoichiometric complex with "partner" HPG or plasmin (HPN), which then catalytically activates free "substrate" molecules of HPG to HPN by selective cleavage of the Arg 561 -Val 562 peptide bond (2, 3). It is believed that consequent to the initial SK⅐HPG complexation, there is a structural rearrangement within the complex, and even before any proteolytic cleavage takes place, an active center within the HPG moiety capable of undergoing acylation is formed (3). This activated complex is rapidly transformed into an SK⅐HPN complex and develops an HPG activator activity. Unlike free HPN, however, which is essentially a trypsin-like protease with broad substrate preference, SK⅐HPN displays a very narrow substrate specificity (4). The structural basis of the conversion of the broadly specific serine protease HPN to a highly substrate-specific protease, once complexed with the "cofactor" SK, with exclusiv...

Section: Resultsmentioning

confidence: 99%

Involvement of a Nine-residue Loop of Streptokinase in the Generation of Macromolecular Substrate Specificity by the Activator Complex through Interaction with Substrate Kringle Domains

Dhar¹,

Pande²,

Sundram

et al. 2002

“…Studies demonstrating a critical role of the N terminus of SK in conformational activation support the "molecular sexuality" mechanism (34,56,57). In this mechanism, insertion of the SK N terminus into the N-terminal binding cleft in the Pg catalytic domain and salt bridge formation with Asp 194 (chymotrypsin numbering) triggers the activating conformational change.…”

Section: Discussionmentioning

confidence: 96%

Resolution of Conformational Activation in the Kinetic Mechanism of Plasminogen Activation by Streptokinase

Boxrud¹,

Verhamme

Bock

2004

Streptokinase (SK) 1 is used clinically as a thrombolytic drug to activate plasminogen (Pg) to plasmin (Pm), the serine proteinase responsible for dissolution of fibrin clots (1). Native [Glu]Pg is a multidomain zymogen consisting of a 77-residue N-terminal peptide, five kringle domains, and a serine proteinase catalytic domain that is activated by cleavage of Arg 561 -Val 562 (2, 3).[Glu]Pg is in a compact conformation, maintained by intramolecular interactions between the N-terminal peptide and lysine-binding sites in kringles 4 and 5 (4 -6). Release of the N-terminal peptide by Pm cleavage generates [Lys]Pg, which adopts an extended conformation and is cleaved by plasminogen activators at a faster rate (5, 7-13). SK possesses no intrinsic catalytic activity but interacts with Pg and Pm, converting both the zymogen and active proteinase into specific proteolytic Pg activators (14 -19). SK binding to Pg results in conformational expression of an active catalytic site on the zymogen without the usual strict requirement for peptide bond cleavage (14,16,17). Pm is generated subsequently by proteolytic cleavage of Arg 561 -Val 562 , and the SK⅐Pm complex propagates Pg activation through expression of a substrate recognition exosite (20,21).It is well established that both conformational and proteolytic activation contribute to SK-induced Pg activation, but there are a number of unresolved questions concerning the mechanism of conformational activation and its coupling to subsequent proteolytic Pm formation. Early studies (14,16,17) demonstrated that interaction of SK with Pg produced the activated Pg catalytic site in the SK⅐Pg* complex. Subsequent kinetic studies indicated that Pg activation involved an initially formed SK⅐Pg* activation complex and an isomerized form of the complex (22, 23). The isomerization was time-, chloride ion-, and fibrinogen-dependent, and the active complexes interconverted slowly under certain experimental conditions. In other studies, a species of Pg isolated from a mixture of SK and Pg was reported to be an active "virgin enzyme" form of free Pg*, suggesting irreversible conformational activation (24). The relationship between SK-Pg binding and conformational activation and its reversibility have not been clearly established.Previous studies of the mechanism of Pg activation by SK have not taken into account the binding affinities of SK for Pg and Pm species, primarily because consistent and reliable equilibrium binding constants have not been available. A number of studies have examined the binding interactions by various experimental approaches but have resulted in a very broad range of dissociation constants, varying from 28 pM to 220 nM for [Glu]Pg (25-32), for example, where the higher affinities may result from Pm generation in SK/Pg mixtures. Our equilibrium binding studies have employed active site-labeled fluorescent Pg and Pm analogs to quantitate SK binding in the absence of proteolytic reactions (21,28,33,34). The results for the active site-labeled proteins support a rela...

“…However, in the presence of plasmin, SAK and SK mechanisms are indistinguishable (12). In the present experiments, active site generation by SUPA was examined under classical experimental conditions where excess acylating agent was present to inhibit trace amounts of plasmin (7,8).…”

Section: Discussionmentioning

confidence: 99%

“…By analogy to the activation of tissue Pg activator, which has significantly greater activity as a zymogen than Pg, it has been suggested that the binding of ␥-domain to the autolysis loop of Pg induces the formation of a critical intramolecular salt bridge between Lys 698 and Asp 740 of Pg that productively structures the activation domain (11). Another hypothesis ("molecular sexuality") states that SK nonproteolytically activates Pg when the NH 2 -terminal isoleucine-1 (Ile 1 ) of the ␣-domain forms a salt bridge with Asp 740 of Pg (12). In this activation sequence, which is typical of the zymogens of the chymotrypsinogen family, salt bridge formation productively restructures the latent activation domain (14).…”

mentioning

confidence: 99%

The Mechanism of a Bacterial Plasminogen Activator Intermediate between Streptokinase and Staphylokinase

Sazonova

Houng

Chowdhry

et al. 2001

Self Cite

The therapeutic properties of plasminogen activators are dictated by their mechanism of action. Unlike staphylokinase, a single domain protein, streptokinase, a 3-domain (␣, ␤, and ␥) molecule, nonproteolytically activates human (h)-plasminogen and protects plasmin from inactivation by ␣ 2 -antiplasmin. Because a streptokinase-like mechanism was hypothesized to require the streptokinase ␥؊domain, we examined the mechanism of action of a novel two-domain (␣,␤) Streptococcus uberis plasminogen activator (SUPA). Under conditions that quench trace plasmin, SUPA nonproteolytically generated an active site in bovine (b)-plasminogen. SUPA also competitively inhibited the inactivation of plasmin by ␣ 2 -antiplasmin. Still, the lag phase in active site generation and plasminogen activation by SUPA was at least 5-fold longer than that of streptokinase. Recombinant streptokinase ␥-domain bound to the b-plasminogen⅐SUPA complex and significantly reduced these lag phases. The SUPA-b⅐plasmin complex activated b-plasminogen with kinetic parameters comparable to those of streptokinase for h-plasminogen. The SUPA-b⅐plasmin complex also activated h-plasminogen but with a lower k cat (25-fold) and k cat /K m (7.9-fold) than SK. We conclude that a ␥-domain is not required for a streptokinase-like activation of b-plasminogen. However, the streptokinase ␥-domain enhances the rates of active site formation in b-plasminogen and this enhancing effect may be required for efficient activation of plasminogen from other species.