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

Lee Fong Lin, Saravuth Oeun, Aiilyan Houng, Guy Reed

Research output: Contribution to journalArticle

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Abstract

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 109 vs (2.4 ± 0.2) x 109 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.

Original languageEnglish (US)
Pages (from-to)16879-16885
Number of pages7
JournalBiochemistry
Volume35
Issue number51
DOIs
StatePublished - Dec 24 1996

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Streptokinase
Plasminogen
Lysine
Mutation
Plasminogen Activators
Molecules
Substrates
Kinetic parameters
Assays
Activator Appliances
Processing
Chemical activation

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Mutation of lysines in a plasminogen binding region of streptokinase identifies residues important for generating a functional activator complex. / Lin, Lee Fong; Oeun, Saravuth; Houng, Aiilyan; Reed, Guy.

In: Biochemistry, Vol. 35, No. 51, 24.12.1996, p. 16879-16885.

Research output: Contribution to journalArticle

Lin, Lee Fong ; Oeun, Saravuth ; Houng, Aiilyan ; Reed, Guy. / Mutation of lysines in a plasminogen binding region of streptokinase identifies residues important for generating a functional activator complex. In: Biochemistry. 1996 ; Vol. 35, No. 51. pp. 16879-16885.
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abstract = "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 109 vs (2.4 ± 0.2) x 109 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.",
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