Mutation of lysines in a plasminogen binding region of streptokinase identifies residues important for generating a functional activator complex

作者：Lin LF; Oeun S; Houng A; Reed GL

来源：Biochemistry, 1996, 35(51): 16879-16885.

DOI：10.1021/bi961531w

Through a unique but poorly understood mechanism, streptokinase (SK) interacts with human plasminogen to generate an ''activator complex'' that efficiently cleaves substrate plasminogen molecules. Previous studies have suggested that lysine residues in SK may play a role in the binding and function of the activator complex. To investigate this hypothesis, 10 different lysine residues in the plasminogen binding region of SK were altered to construct 8 recombinant (r) SK mutants. Only one double mutant, rSK(K256,257A) (replacing Lys with Ala at residues 256 and 257), showed a statistically significant reduction (63%) in binding affinity for Glu-plasminogen. This mutant also displayed a lagtime in the appearance of maximal activity, and modest impairments (2-5-fold) in kinetic parameters for amidolytic and plasminogen activator activity compared to rSK. In contrast, another mutant, rSK(K332,334A), formed an activator complex with profound and nearly selective defects in the catalytic processing of substrate plasminogen molecules. When compared to rSK in kinetic assays of plasminogen activation, the rSK(K332,334A) mutant formed an activator complex that bound substrate plasminogens normally (normal K-m), but its ability to activate or cleave these molecules (k(cat)) was reduced by 34-fold. In contrast, in amidolytic assays, the kinetic parameters of rSK(K332,334A) showed only minor differences (< 2-fold) from rSK. Similarly, the binding affinity of this 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 plasminogen binding region of SK which appear to be necessary for normal high-affinity binding to plasminogen, and for the efficient catalytic processing of substrate plasminogen molecules by the activator complex.

• 出版日期1996-12-24