Journal
ANALYTICAL BIOCHEMISTRY
Volume 342, Issue 1, Pages 20-27Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2005.04.014
Keywords
papain; cathepsin B; benzoxazole; fluorogenic peptide; protease substrate
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New peptide substrates containing benzoxazol-5-yl-alanine derivatives for kinetic assay of cysteine proteases have been synthesized and characterized. The substrates are peptides internally quenched by the intramolecular fluorescence resonance energy transfer. The results demonstrate that the kind of donor-acceptor pair (D-A) significantly affects the kinetic parameters of the enzymatic process. The three longest peptides, Box- Lys-Phe-Gly-Gly-Ala-Ala-Tyr(NO2,) containing Box-alanine derivative as a donor and nitro-tyrosine as an acceptor, show two times greater affinity to papain than does the one peptide possessing Dabcyl-Edans as a D-A pair. Kinetic parameters for the best papain substrate, Lys- B ox(benzfur) - Gly-Gly-Ala-Ala-Tyr(NO2), are K-m = 6.85 +/- 0.59 mu M, k(cat) = 19.51 s(-1), and k(cat)/K-m = 2.85 mu M-1 s(-1). It was found that the peptides Box(benzfur)-Lys-Phe-GlyGly-Tyr(NO,) and Box(benzfur)-Phe-Gly-Gly-Tyr(NO2) were also hydrolyzed by cathepsin B with the highest speed of hydrolysis as a result of caboxypeptidase activity of this enzyme. Moreover, these substrates show high affinity and selectivity to this enzyme. (c) 2005 Elsevier Inc. All rights reserved.
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