Conversion of the zymogen plasminogen (Pg) to the active enzyme plasmin is catalyzed by proteinases such as tissue-type plasminogen activator (t-PA). Interaction of Pg with small ligands such as lysine or macromolecular ligands such as fibrin induces a dramatic conformational change in the zymogen which enhances its efficacy as a t-PA substrate, thereby increasing catalytic efficiency of the activation reaction. We have previously demonstrated that a synthetic peptide derived from amino acids 2091-2108 of the laminin A chain (designated LamA2091-2108) can significantly enhance t-PA-catalyzed Pg activation. To probe the mechanism of this stimulatory reaction, we have determined the effect of substituted LamA2091-2108 derivatives on Pg activation by t-PA. Substitution of charged residues in LamA2091-2108 with neutral amino acids decreases the kcat/Km observed in the presence of native LamA2091-2108. Furthermore, fluorescence-quenching experiments demonstrate that whereas LamA2091-2108 alters the solvent accessibility of Pg Trp residues, charge-substituted peptides have little effect on Pg conformation. These data suggest that LamA2091-2108 stimulates Pg activation by inducing a conformational change in the zymogen similar to that observed upon binding of other ligands such as lysine and fibrin.