Molucular studies on N-terminal domain of streptokinase

Abstract

Streptokinase (SK), an extracellular protein of hemolytic streptococci, is known to lyse blood clots by activating plasminogen to plasmin. SK activates plasminogen by the formation of 1:1 molar complex of SK-plasminogen, which serves as the activator of free plasminogen to form plasmin. It has been suggested that the N-terminal region of SK is important in SK function and that several residues in the N-terminal region are unrelated to plasminogen activation. To investigate the functional role of the N-terminal region of SK, three deletion mutants, in which 7 (SKΔN7), 13 (SKΔN13) and 20 (SKΔN20) amino acids from the N-terminus of SK were truncated, were generated. The purified SKΔN7 and SKΔN13 mutants were nearly as active as wild-type in plasminogen activation. However, the SKΔN20 mutant showed only 4% of specific activity compared to the wild-type SK. In equimolar mixture of plasminogen with SKΔN20, the appearance of maximal amidolytic activity for the SKΔN20-plasminogen complex was observed after incubation for 4 min. The maximal amidolytic activity was not delayed compared to that of wild-type SK, indirectly showing that the deletion of 20 amino acid residues from the SK N-terminus did not reduce the binding affinity to plasminogen. To identify the individual amino acid residues of SK that induce activator function in plasminogen, ten site-directed mutations, S16A, S16L, Q17A, Q17L, L18A, L18Q, V19A, V19Q, V20A and V20Q, were introduced at the region of SK, Ser16-Val20. The data showed that eight SK mutants involving positions 16 (S16A and S16L), 17 (Q17A and Q17L), 18 (L18A and L18Q) and 20 (V20A and V20Q) were nearly as active as the wild-type SK in plasminogen activation. However, the V19A and V19Q mutants showed about 50% and 30% of plasminogen activation compared to wild-type SK, respectively. Three site-directed mutations, V19L, V19D and V19H were newly obtained to characterize the functional role of Val19 in plasminogen activation. The plasminogen-act...