Enzymatic action of prostate-specific antigen (PSA or hk3) : Substrate specificity and regulation by Zn2+, a tight-binding inhibitor

BACKGROUND In semen, prostate-specific antigen (PSA or hK3) digests the gel proteins semenogelin I and II, resulting in liquefaction and the release of motile spermatozoa. We characterized the substrate specificity and zinc-mediated inhibition of PSA. METHODS The proteolysis of human semenogelin I (SgI) and II (SgII) by PSA was characterized by purification of generated SgI and SgII fragments, N-terminal sequencing, and mass spectrometry. Zn2+-inhibition of PSA was studied using a chromogenic substrate. RESULTS Eighteen cleavage sites in SgI and 16 in SgII were identified. Cleavages were identified mainly as the C-terminal of certain tyrosine and glutamine residues, but also the C-terminal of histidine, aspartic acid, leucine, serine, and asparagine residues. No cleavages were identified at any arginine, lysine, phenylalanine, tryptophan, or methionine residues, indicating that the substrate specificity of PSA is distinct from that of trypsin, chymotrypsin, tissue kallkrein (hK1), and kallikrein 2 (hK2). Zn2+ ions have a dramatic effect on PSA activity; the data indicate that Zn2+ is a tight-binding inhibitor of PSA activity. CONCLUSIONS The data will enable the optimized design of PSA activity assays, which may prove instrumental to uncovering the role of PSA in cancer and reproduction. The inhibition data indicate that Zn2+ could regulate PSA activity, which may prove important in the development of efficient inhibitors of PSA activity. Prostate 45:132–139, 2000. © 2000 Wiley-Liss, Inc.