Role of the amino-terminal region of streptokinase in the generation of a fully functional plasminogen activator complex probed with synthetic peptides.
The 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 SK16-59 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.
Nihalani
D
D
Institute of Microbial Technology, Chandigarh, India.
Kumar
R
R
Rajagopal
K
K
Sahni
G
G
D000581:Amidohydrolases / Q000737:chemistry; D004789:Enzyme Activation; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D010455:Peptides / Q000737:chemistry; D010960:Plasminogen Activators / Q000737:chemistry*; D011485:Protein Binding; D011994:Recombinant Proteins; D013300:Streptokinase / Q000737:chemistry*; D013329:Structure-Activity Relationship
D046911:Macromolecular Substances; D010455:Peptides; D011994:Recombinant Proteins; D013300:Streptokinase; D010960:Plasminogen Activators; D000581:Amidohydrolases