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...

To explore the interdomain co-operativity during human plasminogen (HPG) activation by streptokinase (SK), we expressed the cDNAs corresponding to each SK domain individually (␣, ␤, and ␥), and also their twodomain combinations, viz. ␣␤ and ␤␥ in Escherichia coli. After purification, ␣ and ␤ showed activator activities of approximately 0.4 and 0.05%, respectively, as compared with that of native SK, measured in the presence of human plasmin, but the bi-domain constructs ␣␤ and ␤␥ showed much higher co-factor activities (3.5 and 0.7% of native SK, respectively). Resonant Mirror-based binding studies showed that the single-domain constructs had significantly lower affinities for "partner" HPG, whereas the affinities of the two-domain constructs were remarkably native-like with regards to both binary-mode as well as ternary mode ("substrate") binding with HPG, suggesting that the vast difference in cofactor activity between the two-and three-domain structures did not arise merely from affinity differences between activator species and HPG. Remarkably, when the co-factor activities of the various constructs were measured with microplasminogen, the nearly 50-fold difference in the co-factor activity between the two-and three-domain SK constructs observed with full-length HPG as substrate was found to be dramatically attenuated, with all three types of constructs now exhibiting a low activity of approximately 1-2% compared to that of SK⅐HPN and HPG. Thus, the docking of substrate through the catalytic domain at the active site of SKplasmin(ogen) is capable of engendering, at best, only a minimal level of co-factor activity in SK⅐HPN. Therefore, apart from conferring additional substrate affinity through kringle-mediated interactions, reported earlier (Dhar et al., 2002; J. Biol. Chem. 277, 13257), selective interactions between all three domains of SK and the kringle domains of substrate vastly accelerate the plasminogen activation reaction to near native levels. Streptokinase (SK)1 is a widely used bacterial thrombolytic protein that is secreted by several species of ␤-hemolytic streptococci (1, 2). It consists of a single polypeptide chain of 414 residues and is organized into three structurally similar, independently folding domains (termed ␣, ␤, and ␥ in order from N to C terminus of the polypeptide) that are separated by coiled coils and small flexible regions at the two ends (3-5). Like several other well known thrombolytic proteins, such as urokinase and tissue-plasminogen activator, SK exerts its effects through the conversion of human plasminogen (HPG) to its proteolytically active form, plasmin (HPN). Thus, during the treatment of various circulatory disorders, e.g. myocardial infarction, deep vein thrombosis, pulmonary embolism, etc., HPN generated by the activation of HPG helps restore blood flow to the afflicted part by proteolytic dissolution of the fibrin in the pathological clot. In contrast to tissue-plasminogen activator and urokinase, which are intrinsically HPG-specific proteases and thus "directly...

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