Arginine 719 in human plasminogen mediates formation of the staphylokinase:plasmin activator complex.
Staphylokinase (Sak), a 16-kDa bacterial protein, forms a 1:1 stoichiometric complex with the serine proteinase domain of human plasmin, which in turn converts other plasminogen molecules into plasmin. To identify amino acid residues critical for generating the Sak:plasmin activator complex, alanine-scanning mutagenesis was performed on phage-displayed micro-plasminogen (microPlg). Substitution of Arg719 with Ala [microPlg(R719A)] disrupted complex formation, although the sensitivity of phage-displayed microPlg(R719A) to activation by urokinase and the amidolytic activity of the micro-plasmin derivative [microPli(R719A)] remained unaffected. Likewise, the soluble microPlg(R719A) molecule did not generate a functional activator complex with Sak, whereas quantitative activation into plasmin was obtained upon incubation with either urokinase or the Sak:plasmin complex. Real-time biospecific affinity measurements revealed that the Arg --> Ala substitution at position 719 increased the equilibrium dissociation constant between microPlg(R719A) and Sak from 46 nM to 1 microM, primarily by reducing the association rate constant. Arg719 has recently also been implied in the functional complex formation between human plasmin and streptokinase [Dawson, K. M., Marshall, J. M., Raper, R. H., Gilbert, R. J., and Ponting, C. P. (1994) Biochemistry 33, 12042-12047.], suggesting that both bacterial cofactors may share common structural and/or mechanistic aspects for plasminogen activation.
Jespers
L
L
Center for Transgene Technology and Gene Therapy, Flanders Interuniversity Institute for Biotechnology, and Center for Molecular and Vascular Biology, KULeuven, Leuven, Belgium. laurent.jespers@med.kuleuven.ac.be
Van Herzeele
N
N
Lijnen
H R
HR
Van Hoef
B
B
De Maeyer
M
M
Collen
D
D
Lasters
I
I
D019943:Amino Acid Substitution; D001120:Arginine / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D004789:Enzyme Activation; D006801:Humans; D007700:Kinetics; D008666:Metalloendopeptidases / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010958:Plasminogen / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D011994:Recombinant Proteins; D010958:Plasminogen; D001120:Arginine; D008666:Metalloendopeptidases; C022653:auR protein, Staphylococcus aureus