STOPPED-FLOW FLUORESCENCE KINETIC-STUDIES OF GLU-PLASMINOGEN - CONFORMATIONAL-CHANGES TRIGGERED BY AH-SITE LIGAND-BINDING

Binding of 6-aminohexanoic acid to the AH-site, a weak lysine binding site in Glu-plasminogen, alters the conformation of the molecule. The kinetics of the binding and the accompanying conformational change are investigated at pH 7.8, 25-degrees-C. Changes of intrinsic protein fluorescence were measured as a function of time after rapid mixing in a stopped-flow apparatus. The results reflect a two-step reaction mechanism: Rapid association of Glu-plasminogen and 6-aminohexanoic acid (K1 = 44 mM) followed by the conformational change (k2 = 69 s-1 and k-2 = 3 s-1) with an overall dissociation constant K(d) = 2.0 mM. Thus the conformational change is rather fast, t1/2 = 0.01 s. Its importance for the rates of Glu-plasminogen activation reactions is discussed.