Structure-function relationships in staphylokinase as revealed by "clustered charge to alanine" mutagenesis.
Eighteen mutants of recombinant staphylokinase (SakSTAR) in which clusters of two or three charged residues were converted to alanine ("clustered 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 (SakSTAR67) had specific activities of 3%. SakSTAR13 had an intact affinity for plasminogen and a normal rate of active site exposure in equimolar 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 plasmin-SakSTAR13. SakSTAR48 and SakSTAR67 had a 10-20-fold reduced affinity for plasminogen and a markedly reduced active site exposure; their complexes with plasmin had a more than 20-fold reduced catalytic efficiency toward plasminogen. Thus, plasminogen activation by catalytic amounts of SakSTAR is dependent on complex formation between plasmin(ogen) 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. These findings support a mechanism for the activation of plasminogen by SakSTAR involving formation of an equimolar complex of SakSTAR with traces of plasmin, which converts plasminogen to plasmin and, more rapidly, inactive plasminogen-SakSTAR to plasmin-SakSTAR.
Silence
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Center for Molecular and Vascular Biology, University of Leuven, Belgium.
Hartmann
M
M
Gührs
K H
KH
Gase
A
A
Schlott
B
B
Collen
D
D
Lijnen
H R
HR
D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004563:Electrochemistry; D004795:Enzyme Stability; D005341:Fibrinolysin / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008666:Metalloendopeptidases / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010958:Plasminogen / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013211:Staphylococcus aureus / Q000201:enzymology / Q000235:genetics; D013329:Structure-Activity Relationship
D017931:DNA Primers; D004269:DNA, Bacterial; D011994:Recombinant Proteins; D010958:Plasminogen; D005341:Fibrinolysin; D008666:Metalloendopeptidases; C022653:auR protein, Staphylococcus aureus; D000409:Alanine