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|Title:||S3 to S3 ' subsite specificity of recombinant human cathepsin K and development of selective internally quenched fluorescent substrates|
|Authors:||Alves, Marcio FM|
Cotrin, Simone Silva [UNIFESP]
Juliano, Maria Aparecida [UNIFESP]
Juliano, Luiz [UNIFESP]
Carmona, Adriana Karaoglanovic [UNIFESP]
Universidade Federal de São Paulo (UNIFESP)
CUNY Mt Sinai Sch Med
|Citation:||Biochemical Journal. London: Portland Press, v. 373, p. 981-986, 2003.|
|Abstract:||We have systematically examined the S3 to S3' subsite substrate specificity requirements of cathepsin K using internally quenched fluorescent peptides derived from the lead sequence Abz-KLRFSKQ-EDDnp [where Abz is o-aminobenzoic acid and EDDnp is N-(2,4-dinitrophenyl)ethylenediamine]. We assayed six series of peptides, in which each position except Gln was substituted with various natural amino acids. the results indicated that the S3-S1 subsite requirements are more restricted than those of S1'-S3'. Cathepsin K preferentially accommodates hydrophobic amino acids with aliphatic side chains (Leu, Ile and Val) in the S2 site. Modifications at P1 residues also have a large influence on cathepsin K activity. Positively charged residues (Arg and Lys) represent the best accepted amino acids in this position, although a particular preference for Gly was found as well. Subsite S3 accepted preferentially basic amino acids such as Lys and Arg. A broad range of amino acids was accommodated in the remaining subsites. We further explored the acceptance of a Pro residue in the P2 position by cathepsin K in order to develop specific substrates for the enzyme. Two series of peptides with the general sequences Abz-KXPGSKQ-EDDnp and Abz-KPXGSKQ-EDDnp (where X denotes the position of the amino acid that is altered) were synthesized. the substrates Abz-KPRGSKQ-EDDnp and Abz-KKPGSKQ-EDDnp were cleaved by cathepsin K at the Arg-Gly and Gly-Ser bonds respectively, and have been shown to be specific for cathepsin K when compared with other lysosomal cysteine proteases such as cathepsins L and B and with the aspartyl protease cathepsin D.|
|Appears in Collections:||Artigo|
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