Functional properties of recombinant staphylokinase variants obtained by site-specific mutagenesis of methionine-26.
Variants of recombinant staphylokinase (Sak) were produced by site-specific mutagenesis of the unique Met-26 residue and purified to homogeneity from the cell extract of transformed E. coli. The desired mutations were confirmed by cDNA and amino-acid sequence analysis. Sak-M26L, Sak-M26C, Sak-M26R, Sak-M26V and Sak-M26A were selected for further analysis on the basis of their plasminogen activating activity. The specific fibrinolytic activities of Sak-M26L, Sak-M26C and Sak were comparable (76,000 +/- 10,000, 75,000 +/- 2400 and 78,000 +/- 9700 HU/mg, respectively; mean +/- S.E., n = 3 or 4). Active site exposure in equimolar (4.5 microM) mixtures plasminogen at room temperature was more rapid with Sak-M26L than with Sak (quantitative exposure within 4 min and 8 min, respectively). Activation of 1 microM plasminogen by catalytic amounts (5 nM) of Sak-M26L initially appeared to be somewhat faster, but comparable 50 to 60% activation was obtained within 30 min. In contrast, Sak-M26R and Sak-M26V were virtually inactive, did not form active complexes with plasminogen and did not activate plasminogen. The catalytic efficiencies for plasminogen activation were comparable for plasmin-Sak-M26L, plasmin-Sak-M26C and plasmin-Sak (0.14 microM-1 s-1, 0.16 microM-1 s-1 or 0.12 microM-1 s-1, respectively). Comparable dose-dependent lysis of 0.06 ml 125I-fibrin labeled human plasma clots submerged in 0.3 ml human plasma was obtained with Sak-M26L, Sak-M26C and Sak (concentration required for 50% lysis in 2 h, EC50, of 17 +/- 1.6 nM, 19 +/- 1.4 nM and 14 +/- 2.5 nM, respectively), whereas Sak-M26R or Sak-M26V were inactive. Sak-M26A did not form a stable complex with plasminogen, as shown by gel filtration. These data establish that substitution of the unique Met residue in position 26 of the Sak sequence with Leu or Cys has little or no influence on its plasminogen activating or fibrinolytic potential. In contrast, substitution of Met-26 with either Arg or Val results in total loss of the functional activity. Thus, the amino acid in position 26 of Sak appears to be of crucial importance for the activation of plasminogen by staphylokinase.
Schlott
B
B
Institut für Molekulare Biotechnologie, Jena, Germany.
Hartmann
M
M
Gührs
K H
KH
Birch-Hirschfeld
E
E
Gase
A
A
Vettermann
S
S
Collen
D
D
Lijnen
H R
HR
D000595:Amino Acid Sequence; D001665:Binding Sites; D008666:Metalloendopeptidases / Q000235:genetics / Q000378:metabolism*; D008715:Methionine; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010958:Plasminogen / Q000378:metabolism; D010960:Plasminogen Activators / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
D011994:Recombinant Proteins; D010958:Plasminogen; D008715:Methionine; D010960:Plasminogen Activators; D008666:Metalloendopeptidases; C022653:auR protein, Staphylococcus aureus