Design of new and sensitive fluorogenic substrates for human kallikrein hK3 (prostate-specific antigen) derived from semenogelin sequences.
Human kallikrein hK3 (prostate-specific antigen) is a chymotrypsin-like serine protease which is widely used in the diagnosis of prostate cancer. Assays of the enzymatic activity of hK3 in extracellular fluids have been limited by a lack of sensitive synthetic substrates. This report describes the design of a series of internally quenched fluorescent peptides containing an amino acid sequence based on preferential hK3 cleavage sites in semenogelins. Those were identified by 2-D gel electrophoresis analysis and N-terminal sequencing of semenogelin fragments generated by ex vivo proteolysis in freshly ejaculated semen. These peptides were cleaved by hK3 at the C-terminal of certain tyrosyl or glutaminyl residues with k(cat)/K(m) values of 15000-60000 M(-1) s(-1). The substrate Abz-SSIYSQTEEQ-EDDnp was cleaved at the Tyr-Ser bond with a specificity constant k(cat)/K(m) of 60000 M(-1) s(-1), making it the best substrate for hK3 described to date.
Réhault
Sophie
S
Laboratory of Enzymology and Protein Chemistry, INSERM EMI-U 00-10, University François Rabelais, 2bis Boulevard Tonnellé, 37032 Tours Cedex, France.
Brillard-Bourdet
Michèle
M
Bourgeois
Luc
L
Frenette
Gilles
G
Juliano
Luiz
L
Gauthier
Francis
F
Moreau
Thierry
T
D001665:Binding Sites; D015195:Drug Design; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D008297:Male; D017430:Prostate-Specific Antigen / Q000032:analysis / Q000737:chemistry*; D012661:Semen / Q000737:chemistry / Q000502:physiology*; D029607:Seminal Vesicle Secretory Proteins / Q000737:chemistry*; D013379:Substrate Specificity
D029607:Seminal Vesicle Secretory Proteins; C424482:seminal vesicle-specific antigen; D017430:Prostate-Specific Antigen