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 D stems from the search for specific therapeutic agents against many of the sequentially and structurally homologous members of the 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-ligand 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 rule-based 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 kinetic studies. 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.
Scarborough
P E
PE
Department of Biochemistry and Molecular Biology, University of Florida, Gainesville 32610-0245.
Dunn
B M
BM
D000595:Amino Acid Sequence; D016282:Aspartic Acid Endopeptidases / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D002402:Cathepsin D / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005818:Genetic Engineering; D005971:Glutamates / Q000235:genetics; D018698:Glutamic Acid; D005973:Glutamine / Q000235:genetics; D006801:Humans; D008099:Liver / Q000201:enzymology; D008715:Methionine / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010436:Pepstatins / Q000737:chemistry; D016133:Polymerase Chain Reaction; D017510:Protein Folding; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity
D005971:Glutamates; D010436:Pepstatins; D011994:Recombinant Proteins; D005973:Glutamine; C014195:Streptomyces pepsin inhibitor; D018698:Glutamic Acid; D008715:Methionine; D016282:Aspartic Acid Endopeptidases; D002402:Cathepsin D; C031375:pepstatin