MUTATION OF LYSINES IN A PLASMINOGEN BINDING REGION OF STREPTOKINASE IDENTIFIES RESIDUES IMPORTANT FOR GENERATING A FUNCTIONAL ACTIVATOR COMPLEX

Through a unique but poorly understood mechanism, streptokinase (SK) i nteracts with human plasminogen to generate an ''activator complex'' t hat efficiently cleaves substrate plasminogen molecules. Previous stud ies have suggested that lysine residues in SK may play a role in the b inding and function of the activator complex. To investigate this hypo thesis, 10 different lysine residues in the plasminogen binding region of SK were altered to construct 8 recombinant (r) SK mutants. Only on e double mutant, rSK(K256,257A) (replacing Lys with Ala at residues 25 6 and 257), showed a statistically significant reduction (63%) in bind ing affinity for Glu-plasminogen. This mutant also displayed a lagtime in the appearance of maximal activity, and modest impairments (2-5-fo ld) in kinetic parameters for amidolytic and plasminogen activator act ivity compared to rSK. In contrast, another mutant, rSK(K332,334A), fo rmed an activator complex with profound and nearly selective defects i n the catalytic processing of substrate plasminogen molecules. When co mpared to rSK in kinetic assays of plasminogen activation, the rSK(K33 2,334A) mutant formed an activator complex that bound substrate plasmi nogens normally (normal K-m), but its ability to activate or cleave th ese molecules (k(cat)) was reduced by 34-fold. In contrast, in amidoly tic assays, the kinetic parameters of rSK(K332,334A) showed only minor differences (< 2-fold) from rSK. Similarly, the binding affinity of t his mutant to human Glu-plasminogen was indistinguishable from rSK [(2 .6 +/- 0.8) x 10(9) vs (2.4 +/- 0.2) x 10(9) M(-1), respectively]. In summary, these experiments have identified lysine residues in a plasmi nogen binding region of SK which appear to be necessary for normal hig h-affinity binding to plasminogen, and for the efficient catalytic pro cessing of substrate plasminogen molecules by the activator complex.