Plasminogen activator inhibitor 1 (PAI-1), a member of the serpins, has a unique conformational flexibility. A typical characteristic is its intrinsic lability resulting in the conversion of the active conformation to a latent conformation, In the present study, we have evaluated the effect of substitution of positively charged residues located at the turn connecting strand s4C with strand s3C, either with negatively charged or with neutral residues, on the functional stability of PAI-1. The following mutants were constructed, purified and characterized in comparison to wild-type (wt) PAI-1: PAT-1-R186E,R187E (Arg(186) --> Glu and Arg(187) --> Glu), PAI-1-H190E,K191E (His(190) --> Glu and Lys(191) --> Glu) and PAI-1-H190L,K191L (His(190) --> Leu and Lys(191) --> Leu). In contrast to wtPAI-1 the mutants exhibited no inhibitory activity, Whereas latent wtPAI-1 can be reactivated (up to a specific activity of 78 +/- 19%) by treatment with guanidinium chloride, a similar treatment applied to these mutants resulted in a significant but relatively small increase of specific activity (i.e. to 14%). Evaluation of the functional stability (at 37 degrees C, pH 5.5, 1 M NaCl) revealed a strongly decreased functional stability compared to wtPAI-1 (i.e. 3-9 h for the mutants vs. > 24 h for wtPAI-1). Further characterization by heat denaturation studies and plasmin susceptibility confirmed that removal or reversal of the positive charge on the turn connecting s4C with s3C results in PAI-1 mutants with a highly accelerated conversion of active to latent forms. We can therefore conclude that the pronounced positive charge in the turn connecting s3C with s3C is of the highest importance for the functional stability of PAI-1. (C) 1997 Federation of European Biochemical Societies.