Stopped-flow fluorescence kinetic studies of Glu-plasminogen Conformational changes triggered by AH-site ligand binding

Abstract

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°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 ( K 1 = 44 mM) followed by the conformational change ( k 2 = 69 g −1 and k −2 = 3 g −1) with on overall dissociation constant K 4 = 2.0 mM, Thus the conformational change is rather fast, t 1 2 = 0.01 g. Its importance for the rates or Glu-plasminogen activation reactions is discussed.