Redesign of the substrate specificity of human cathepsin D: the dominant role of position 287 in the S2 subsite

Interest in the active site specificity of human cathepsin Dstems from the search for specific therapeutic agents againstmany of the sequentially and structurally homologous membersofthe aspartic proteinase family. The work presented here examinedone amino acid in the cathepsin D sequence, located in the S₂subsite, which contributes substantially to the specificityof enzyme-Ugand interactions at the enzyme active site. Previousstudies reported on the specificity of binding and catalysisby native and recombinant human cathepsin D explored throughkinetic studies using a systematic series of synthetic substrates.Utilizing a rulebased molecular model of human cathepsin D,Met287 was suggested as a candidate for mutagenesis to furtherexplore selectivity within the S₂ subsite of the cathepsin Dactive site. Met287 mutant derivatives of human cathepsin Dwere designed, expressed and characterized in kineticstudies.Native cathepsin D accommodates large hydrophobic residues inthe P₂ position of a substrate; positively charged residuesin P₂ are not favorable for catalysis.It was demonstrated thataltering Met287 of human cathepsin D to more polar amino acidsproduced active mutant enzymes with significantly altered substratespecificity.