STRUCTURE-FUNCTION-RELATIONSHIPS IN STAPHYLOKINASE AS REVEALED BY CLUSTERED CHARGE TO ALANINE MUTAGENESIS

Eighteen mutants of recombinant staphylokinase (SakSTAR) in which clus ters of two or three charged residues were converted to alanine (''clu stered charge-to-alanine scan'') were characterized, Fifteen of these mutants had specific plasminogen-activating activities of >20% of that of wild-type SakSTAR, whereas three mutants, SakSTAR K11A D13A D14A ( SakSTAR13), SakSTAR E46A K50A (SakSTAR48), and SakSTAR E65A D69A (SakS TARB67) had specific activities of 3%. SakSTAR13 had an intact affinit y for plasminogen and a normal: rate of active site exposure in equimo lar mixtures with plasminogen. The plasmin-SakSTAR13 complex had a 14- fold reduced catalytic efficiency for plasminogen activation but was 5 -fold more efficient for conversion of plasminogen-SakSTAR13 to plasmi n-SakSTAR13, SakSTAR48 and SakSTAR67 had a 10-20-fold reduced affinity for plasminogen and a markedly re reduced active site exposure; their complexes with plasmin had a more than 20-fold reduced catalytic effi ciency toward plasminogen. Thus, plasminogen activation by catalytic a mounts of SakSTAR is dependent on complex formation between plasmin(og en) and SakSTAR, which is deficient with SakSTAR48 and SakSTAR67, but also on the induction of a functional active site configuration in the plasmin-SakSTAR complex, which is deficient with all three mutants, T hese findings support a mechanism for the activation of plasminogen by SakSTAR involving formation of an equimolar complex of SakSTAR with t races of plasmin, which converts plasminogen to plasmin and, more rapi dly, inactive plasminogen-SakSTAR to plasmin-SakSTAR.