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 of cathepsin L almost 10-fold. After the auto-activation of the 38 kDa procathepsin L at least three processing sites were determined by N-terminal amino acid sequencing. After replacing the Ala205 residue by glutamic acid, cathepsin B-like specificity was introduced into cathepsin L. This mutation resulted 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 thereby confirmed experimentally to be critical for the specificity of cathepsins B and L.
Barlic-Maganja
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D
Department of Biochemistry and Molecular Biology, Jozef Stefan Institute, Ljubljana, Slovenia.
Dolinar
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M
Turk
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V
D000409:Alanine / Q000378:metabolism*; D019943:Amino Acid Substitution; D056668:Cathepsin L; D002403:Cathepsins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003546:Cysteine Endopeptidases; D016264:Dextran Sulfate / Q000494:pharmacology*; D010450:Endopeptidases; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D004792:Enzyme Precursors; D011994:Recombinant Proteins; D016264:Dextran Sulfate; D002403:Cathepsins; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; C063905:procathepsin L; C534281:CTSL protein, human; D056668:Cathepsin L; D000409:Alanine