Substrate specificity of human cathepsin D using internally quenched fluorescent peptides derived from reactive site loop of kallistatin.
Kallistatin, a serpin that specifically inhibits human tissue kallikrein, was demonstrated to be cleaved at the Phe-Phe bond in its reactive site loop (RSL) by cathepsin D. Internally quenched fluorescent peptides containing the amino acid sequence of kallistatin RSL were highly susceptible to hydrolysis by cathepsin D. Surprisingly, these peptides were efficiently hydrolyzed at Phe-Phe bond, despite having Lys and Ser at P2 and P2' positions, respectively, which was reported to be very unfavorable for substrates for cathepsin D. Due to the importance of cathepsin D in several physiological and pathological processes, we took the peptide containing kallistatin RSL sequence, Abz-Ala-Ile-Lys-Phe-Phe-Ser-Arg-Gln-EDDnp, as a reference substrate for a systematic specificity study of S3 to S3' protease subsites (EDDnp=N-[2,4-dinitrophenyl]-ethylenediamine and Abz=ortho-amino benzoic acid). We present in this paper some internally quenched fluorescent peptides that were efficient substrates for cathepsin D. They essentially differ from other previously described substrates by their higher kcat/Km values due, mainly, to low Km values, such as the substrate Abz-Ala-Ile-Ala-Phe-Phe-Ser-Arg-Gln-EDDnp (Km=0.27 microM, kcat=16.25 s(-1), kcat/Km=60185 microM(-1) x s(-1)).
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Department of Biophysics, Escola Paulista de Medicina-NIFESP, Rua Três de Maio, 100, 2o andar, 04044-020, São Paulo, Brazil.
Oliveira
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Juliano
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Juliano
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D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000737:chemistry*; D002402:Cathepsin D / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D010446:Peptide Fragments / Q000378:metabolism*; D015843:Serpins / Q000737:chemistry*; D013379:Substrate Specificity
D002352:Carrier Proteins; D005456:Fluorescent Dyes; D010446:Peptide Fragments; D015843:Serpins; C051324:kallistatin; D002402:Cathepsin D