Positive co-operative binding at two weak lysine-binding sites governs the Glu-plasminogen conformational change.
The kinetics of a series of Glu-plasminogen ligand-binding processes were investigated at pH 7.8 and 25 degrees C (in 0.1 M-NaCl). The ligands include compounds analogous to C-terminal lysine residues and to normal lysine residues. Changes of the Glu-plasminogen protein fluorescence were measured in a stopped-flow instrument as a function of time after rapid mixing of Glu-plasminogen and ligand at various concentrations. Large positive fluorescence changes (approximately 10%) accompany the ligand-induced conformational changes of Glu-plasminogen resulting from binding at weak lysine-binding sites. Detailed studies of the concentration-dependencies of the equilibrium signals and the rate constants of the process induced by various ligands showed the conformational change to involve two sites in a concerted positive co-operative process with three steps: (i) binding of a ligand at a very weak lysine-binding site that preferentially, but not exclusively, binds C-terminal-type lysine ligands, (ii) the rate-determining actual-conformational-change step and (iii) binding of one more lysine ligand at a second weak lysine-binding site that then binds the ligand more tightly. Further, totally independent initial small negative fluorescence changes (approximately 2-4%) corresponding to binding at the strong lysine-binding site of kringle 1 [Sottrup-Jensen, Claeys, Zajdel, Petersen & Magnusson (1978) Prog. Chem. Fibrinolysis Thrombolysis 3, 191-209] were observed for the C-terminal-type ligands. The finding that the conformational change in Glu-plasminogen involves two weak lysine-binding sites indicates that the effect cannot be assigned to any single kringle and that the problem of whether kringle 4 or kringle 5 is responsible for the process resolves itself. Probably kringle 4 and 5 are both participating. The involvement of two lysine binding-sites further makes the high specificity of Glu-plasminogen effectors more conceivable.
Christensen
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U
Department of Chemistry, University of Copenhagen, Denmark.
Mølgaard
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L
D015119:Aminocaproic Acid / Q000378:metabolism; D001665:Binding Sites; D005454:Fluorescence Polarization; D006801:Humans; D007700:Kinetics; D008239:Lysine / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D010958:Plasminogen / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D014148:Tranexamic Acid / Q000378:metabolism
D010446:Peptide Fragments; D014148:Tranexamic Acid; D010958:Plasminogen; D008239:Lysine; D015119:Aminocaproic Acid