Substrate specificity of prostate-specific antigen (PSA).
BACKGROUND
The serine protease prostate-specific antigen (PSA) is a useful clinical marker for prostatic malignancy. PSA is a member of the kallikrein subgroup of the (chymo)trypsin serine protease family, but differs from the prototypical member of this subgroup, tissue kallikrein, in possessing a specificity more similar to that of chymotrypsin than trypsin. We report the use of two strategies, substrate phage display and iterative optimization of natural cleavage sites, to identify labile sequences for PSA cleavage.


RESULTS
Iterative optimization and substrate phage display converged on the amino-acid sequence SS(Y/F)Y decreases S(G/S) as preferred subsite occupancy for PSA. These sequences were cleaved by PSA with catalytic efficiencies as high as 2200-3100 M-1 s-1, compared with values of 2-46 M-1 s-1 for peptides containing likely physiological target sequences of PSA from the protein semenogelin. Substrate residues that bind to secondary (non-S1) subsites have a critical role in defining labile substrates and can even cause otherwise disfavored amino acids to bind in the primary specificity (S1) pocket.


CONCLUSIONS
The importance of secondary subsites in defining both the specificity and efficiency of cleavage suggests that substrate recognition by PSA is mediated by an extended binding site. Elucidation of preferred subsite occupancy allowed refinement of the structural model of PSA and should facilitate the development of more sensitive activity-based assays and the design of potent inhibitors.
Coombs
G S
GS
Corvas International, Department of Molecular Biology, San Diego, CA 92121, USA.
Bergstrom
R C
RC
Pellequer
J L
JL
Baker
S I
SI
Navre
M
M
Smith
M M
MM
Tainer
J A
JA
Madison
E L
EL
Corey
D R
DR
D000595:Amino Acid Sequence; D014408:Biomarkers, Tumor; D002918:Chymotrypsin / Q000378:metabolism; D016384:Consensus Sequence; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000145:classification / Q000378:metabolism; D010449:Peptide Mapping; D017430:Prostate-Specific Antigen / Q000378:metabolism*; D011487:Protein Conformation; D013379:Substrate Specificity; D009626:Terminology as Topic
D014408:Biomarkers, Tumor; D010446:Peptide Fragments; D002918:Chymotrypsin; D017430:Prostate-Specific Antigen