Role of the amino‐terminal region of streptokinase in the generation of a fully functional plasminogen activator complex probed with synthetic peptides
Authors: Nihalani Deepak , Kumarm Rajesh , Rajagopal K. , Sahni Girish 
Published: July 2010

AbstractThe mechanism whereby fragments of streptokinase (SK) derived from its N terminus (e.g., SK1‐59 or SK1‐63) enhance the low plasminogen (PG)‐activating ability of other fragments, namely SK64‐386, SK60‐414, SK60‐387, and SK60‐333 (reported previously), has been investigated using a synthetic peptide approach. The addition of either natural SK1‐59, or chemically synthesized SK16‐59, at saturation (about 500‐fold molar excess) generated amidolytic and PG activation capabilities in equimolar mixtures of human plasminogen (HPG) and its complementary fragment (either SK60‐414 or SK56‐414, prepared by expression of truncated SK gene fragments in Escherichia coli) that were approximately 1.2‐ and 2.5‐fold, respectively, of that generated by equimolar mixtures of native SK and HPG. Although in the absence of SK1‐59 equimolar mixtures of SK56‐414 and HPG could generate almost 80% of amidolytic activity, albeit slowly, less than 2% level of PG activation could be observed under the same conditions, indicating that the contribution of the N‐terminal region lay mainly in imparting in SK56‐414 an enhanced ability for PG activation. The ability of various synthetic peptides derived from the amino‐terminal region (SK16‐51, SK16‐45, SK37‐59, SK1‐36, SK16‐36, and SK37‐51) to (1) complement equimolar mixtures of SK56‐414 and HPG for the generation of amidolytic and PG activation functions, (2) inhibit the potentiation of SK56‐414 and HPG by SK16‐59, and (3) directly inhibit PG activation by the 1:1 SK‐HPG activator complex was tested. Apart from SK16‐59, SK16‐51, and 16‐45, the ability to rapidly generate amidolytic potential in HPG in the presence of SK56‐414 survived even in the smaller SK‐peptides, viz., SK37‐59 and SK37‐51. However, this ability was abolished upon specifically mutating the sequence ‐LTSRP‐, present at position 42‐46 in native SK. Although SK16‐51 retained virtually complete ability for potentiation of PG activation in comparison to SKI 6‐59 or SK1‐59, this ability was reduced by approximately fourfold in the case of SK16‐45, and completely abolished upon further truncation of the C‐terminal residues to SK16‐36 or SK1‐36. Remarkably, however, these peptides not only displayed ability to bind PG, but also showed strong inhibition of PG activation by the native activator complex in the micromolar range of concentration; the observed inhibition, however, could be competitively relieved by increasing the concentration of substrate PG in the reaction, suggesting that this region in SK contains a site directed specifically toward interaction with substrate PG. This conclusion was substantiated by the observation that the potentiation of PG activating ability was found to be considerably reduced in a peptide (SK25‐59) in which the sequence corresponding to this putative locus (residues 16‐36) was truncated at the middle. On the other hand, fragments SK37‐51 and SK37‐59 did not show any inhibition of the PG activation by native activator complex. Taken together, these findings strongly support a model of SK action wherein the HPG binding site resident in the region 37‐51 helps in anchoring the N‐terminal domain to the strong intermolecular complex formed between HPG and the region 60‐414. In contrast, the site located between residues 16 and 36 is qualitatively more similar to the previously reported PG interacting site (SK254‐273) present in the core region of SK, in being involved in the relatively low‐affinity enzyme‐substrate interactions of the activator complex with PG during the catalytic cycle.