Mechanistic and molecular evolutionary studies on streptokinase

Abstract

Random mutagenesis of streptokinase was carried out to determine critical amino acid residues in plasminogen activation. Fourteen streptokinase mutants without plasminogen activation activity on skim milk-plasminogen overlay plate were selected and sequenced. Specific activities of the selected streptokinase mutants were determined by chromogenic assay. Six mutants (D41C, S44K, S44P, R45P, H48T and D220G) showed substantial amidolytic activities as compared with that of wild type. Moreover, five point mutations in Asp41-His48 region of a streptokinase plays an important role in binding to a substrate plasminogen. Because of the structural and sequence similarity of SKα domain to those of staphylokinase, it is thought that they play a similar role. The chimeric proteins substituted SKα domain with staphylokinase were examined for their activity of plasminogen activation. The kinetics of equimolar mixture of a plasminogen and the fusion proteins showed that these fusion proteins bound to human plasmin for plasminogen activation. Fibrinogen degradation product enhanced the activity of staphylokinase-SKβ fused protein suggesting that fibrin specificity of the fusion protein. These results suggest that the fusion proteins activate a human plasminogen in staphylokinase mode and that the domains of streptokinase did not influence the inhibition of human plasmin by α2-antiplasmin. The N-terminus of staphylokinase-streptokinase fusion protein was substituted by the N-terminus of streptokinase. The serial substitution showed that the non-hydrophilic residues at the N-terminus of the fusion proteins without SKγ domain did not affect the formation of active plasmin-activator complex. However, the fusion mutants with γ domain shortened the lag times in kinetic data. These results propose that streptokinase requires the N-terminal non-hydrophilic residues and γ domain for formation of the active streptokinase-plasminogen complex. Recombinant bovine plasminogen activator also...