SUBSTRATE-SPECIFICITY OF PROSTATE-SPECIFIC ANTIGEN (PSA)

Background: The serine protease prostate-specific antigen (PSA) isa us eful 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 kallikre in, in possessing a specificity more similar to that of chymotrypsin t han trypsin. We report the use of two strategies, substrate phage disp lay 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 down arrow S(G/S) as preferred subsite occupancy for PSA. These seque nces were cleaved by PSA with catalytic efficiencies as high as 2200-3 100 M-1 s(-1), compared with values of 2-46 M-1 s(-1) for peptides con taining likely physiological target sequences of PSA from the protein semenogelin. Substrate residues that bind to secondary (non-S1) subsit es have a critical role in defining labile substrates and can even cau se otherwise disfavored amino acids to bind in the primary specificity (S1) pocket. Conclusions: The importance of secondary subsites in def ining both the specificity and efficiency of cleavage suggests that su bstrate recognition by PSA is mediated by an extended binding site. El ucidation of preferred subsite occupancy allowed refinement of the str uctural model of PSA and should facilitate the development of more sen sitive activity-based assays and the design of potent inhibitors.