Binding of streptokinase (SK) to plasminogen (Pg) activates the zymogen conformationally and initiates its conversion into the fibrinolytic proteinase, plasmin (Pm). Equilibrium binding studies of SK interactions with a homologous series of catalytic site-labeled fluorescent Pg and Pm analogues were performed to resolve the contributions of lysine binding site interactions, associated changes between extended and compact conformations of Pg, and activation of the proteinase domain to the affinity for SK. SK bound to fluorescein-labeled [Glu]Pg(1) and [Lys]Pg(1) with dissociation constants of 624 +/- 112 and 38 +/- 5 nM, respectively, whereas labeled [Lys]Pm(1) bound with a 57000-fold tighter dissociation constant of 11 +/- 2 pM. Saturation of lysine binding sites with 6-aminohexanoic acid had no effect on SK binding to labeled [Glu]Pg(1), but weakened binding to labeled [Lys]Pg(1) and [Lys]Pm(1) 31- and 20-fold, respectively. At low Cl(-) concentrations, where [Glu]Pg assumes the extended conformation without occupation of lysine binding sites, a 23-fold increase in the affinity of SK for labeled [Glu]Pg(1) was observed, which was quantitatively accounted for by expression of new lysine binding site interactions. The results support the conclusion that the SK affinity for the fluorescent Pg and Pm analogues is enhanced 13-16-fold by conversion of labeled [Glu]Pg to the extended conformation of the [Lys]Pg derivative as a result of lysine binding site interactions, and is enhanced 3100-3500-fold further by the increased affinity of SK for the activated proteinase domain. The results imply that binding of SK to [Glu]Pg results in transition of [Glu]Pg to an extended conformation in an early event in the SK activation mechanism.