FUNCTIONAL-PROPERTIES OF RECOMBINANT STAPHYLOKINASE VARIANTS OBTAINEDBY SITE-SPECIFIC MUTAGENESIS OF METHIONINE-26

Variants of recombinant staphylokinase (Sak) were produced by site-spe cific mutagenesis of the unique Met-26 residue and purified to homogen eity from the cell extract of transformed E. coli. The desired mutatio ns were confirmed by cDNA and amino-acid sequence analysis. Sak-M26L, Sak-M26C, Sak-M26R, Sak-M26V and Sak-M26A were selected for further an alysis on the basis of their plasminogen activating activity. The spec ific fibrinolytic activities of Sak-M26L, Sak-M26C and Sak were compar able (76000 +/- 10000, 75000 +/- 2400 and 78000 +/- 9700 HU/mg, respec tively; mean +/- S.E., n = 3 or 4). Active site exposure in equimolar (4.5 mu M) mixtures with plasminogen at room temperature was more rapi d with Sak-M26L than with Sak (quantitative exposure within 4 min and 8 min, respectively). Activation of 1 mu M plasminogen by catalytic am ounts (5 nM) of Sak-M26L initially appeared to be somewhat faster, but comparable 50 to 60% activation was obtained within 30 min. In contra st, Sak-M26R and Sak-M26V were virtually inactive, did not form active complexes with plasminogen and did not activate plasminogen. The cata lytic efficiencies for plasminogen activation were comparable for plas min-Sak-M26L, plasmin-Sak-M26C and plasmin-Sak (0.14 mu M(-1) s(-1), 0 .16 mu M(-1) s(-1) or 0.12 mu M(-1) s(-1) respectively). Comparable do se-dependent lysis of 0.06 ml I-125-fibrin labeled human plasma clots submerged in 0.3 mi human plasma was obtained with Sak-M26L, Sak-M26C and Sak (concentration required for 50% lysis in 2 h, EC(50), 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 th at substitution of the unique Met residue in position 26 of the Sak se quence with Leu or Cys has little or no influence on its plasminogen a ctivating or fibrinolytic potential. In contrast, substitution of Met- 26 with either Arg or Val results in total loss of the functional acti vity. Thus, the amino acid in position 26 of Sak appears to be of cruc ial importance for the activation of plasminogen by staphylokinase.