Cathepsin B carboxydipeptidase specificity analysis using internally quenched fluorescent peptides.
We have examined in detail the specificity of the subsites S1, S2, S1' and S2' for the carboxydipeptidase activity of cathepsin B by synthesizing and assaying four series of internally quenched fluorescent peptides based on the sequence Dnp-GFRFW-OH, where Dnp (2,4-dinitrophenyl) is the quenching group of the fluorescence of the tryptophan residue. Each position, except the glycine, was substituted with 15 different naturally occurring amino acids. Based on the results we obtained, we also synthesized efficient and sensitive substrates that contained o -aminobenzoic acid and 3-Dnp-(2,3-diaminopropionic acid), or epsilon-amino-Dnp-Lys, as the fluorescence donor-receptor pair. The higher kinetic parameter values for the carboxydipeptidase compared with the endopeptidase activity of cathepsin B allowed an accurate analysis of its specificity. The subsite S1 accepted preferentially basic amino acids for hydrolysis; however, substrates with phenylalanine and aliphatic side-chain-containing amino acids at P1 had lower K m values. Despite the presence of Glu245 at S2, this subsite presented clear preference for aromatic amino acid residues, and the substrate with a lysine residue at P2 was hydrolysed better than that containing an arginine residue. S1' is essentially a hydrophobic subsite, and S2' has particular preference for phenylalanine or tryptophan residues.
Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de São Paulo, Rua Três de Maio, 100, São Paulo 04044-020, Brazil.
Maria Helena S
MH
Cezari
Luciano
L
Puzer
Maria Aparecida
MA
Juliano
Adriana K
AK
Carmona
Luiz
L
Juliano
D002401:Cathepsin B / Q000378:metabolism*; D004150:Dipeptidases / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity
D005456:Fluorescent Dyes; D010455:Peptides; D004150:Dipeptidases; D002401:Cathepsin B