The influence of Ala205 on the specificity of cathepsin L produced by dextran sulfate assisted activation of the recombinant proenzyme

Human procathepsin L has been expressed in E. coli in the form of inclusion bodies. The recombinant protein was isolated, refolded and processed at pH 5.5 by the addition of dextran sulfate which increased the overall yield o f cathepsin L almost 10-fold. After the auto-activation of the 38 kDa proca thepsin L at least three processing sites were determined by N-terminal ami no acid sequencing. After replacing the Ala205 residue by glutamic acid, ca thepsin B-like specificity was introduced into cathepsin L. This mutation r esulted in a 15-fold increased activity toward the substrate Z-Arg-Arg-AMC and in a 29-fold decreased activity toward Z-Phe-Arg-AMC. Residue 205 is th ereby confirmed experimentally to be critical for the specificity of cathep sins B and L.