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

Abstract

Interest in the active site specificity of human cathepsin D stems from the search for specific therapeutic agents against many of the sequentially and structurally homologous members ofthe aspartic proteinase family. The work presented here examined one amino acid in the cathepsin D sequence, located in the S₂ subsite, which contributes substantially to the specificity of enzyme-Ugand interactions at the enzyme active site. Previous studies reported on the specificity of binding and catalysis by native and recombinant human cathepsin D explored through kinetic 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 further explore selectivity within the S₂ subsite of the cathepsin D active site. Met287 mutant derivatives of human cathepsin D were designed, expressed and characterized in kineticstudies. Native cathepsin D accommodates large hydrophobic residues in the P₂ position of a substrate; positively charged residues in P₂ are not favorable for catalysis.It was demonstrated that altering Met287 of human cathepsin D to more polar amino acids produced active mutant enzymes with significantly altered substrate specificity.