FUNCTIONAL-ANALYSIS OF THE AMINO-TERMINI AND CARBOXYL-TERMINI OF STREPTOKINASE

Streptokinase (SK) is a 414 amino acid bacterial protein that activate s human plasminogen. Streptokinase fragments derived from the central portion of the protein bind plasminogen, but are inactive, indicating that the amino- and/or carboxyl-termini are required for normal plasmi nogen activator activity. To better define the function of the N- and C-termini of SK we generated and characterized 21 N-terminal and 20 C- terminal deletion mutants. All mutants lacking greater than or equal t o 18 N-terminal or greater than or equal to 51 C-terminal amino acids exhibited markedly reduced plasminogen activator activity, while mutan ts lacking less than or equal to 12 N-terminal or less than or equal t o 40 C-terminal residues were fully active. The decrease in SK activit y with N-terminal deletion appeared to result not from loss of plasmin ogen binding capacity, but rather from increased susceptibility of del etion mutants to degradation by plasmin. Point mutations at positions 13 (SK V13D) or 20 (SK V20D) produced functional abnormalities similar to those observed in N-terminal deletion mutants, with SK V13D exhibi ting delayed amidolytic activity and SK V20D exhibiting only 1% plasmi nogen activator activity and marked sensitivity to degradation by plas min. C-terminal deletion mutants lacking greater than or equal to 51 a mino acids also bound plasminogen, but did not induce significant amid olytic activity in plasminogen or activator activity in plasmin. Preve ntion of cleavage at position 59 of SK had no effect on plasminogen ac tivator activity, suggesting that the rapid hydrolysis of this bond th at occurs after SK-plasminogen complex formation is not required for n ormal function of the N-terminus. These results suggest that residues within or near positions 13-20 of SK are important determinants of its capacity to generate amidolytic activity and are a critical determina nt of the stability of SK, while residues within or near position 364- 374 are required for generating amidolytic activity and for conferring plasminogen activator activity to plasmin(ogen). These results also s uggest that SK fragments significantly smaller than SK 13-374 are unli kely to be effective thrombolytic agents.