Enzymatic action of prostate-specific antigen (PSA or hK3): Substrate specificity and regulation by Zn2+, a tight-binding inhibitor

BACKGROUND. Ln semen, prostate-specific antigen (PSA or hK3) digests the ge l 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-te rminal sequencing, and mass spectrometry. Zn2+-inhibition of PSA was studie d using a chromogenic substrate. RESULTS. Eighteen cleavage sites in SgI and 16 in SgII were identified. Cle avages were identified mainly as the C-terminal of certain tyrosine and glu tamine residues, but also the C-terminal of histidine, aspartic acid, leuci ne, serine, and asparagine residues. No cleavages were identified at any ar ginine, lysine, phenylalanine, tryptophan, or methionine residues, indicati ng that the substrate specificity of PSA is distinct from that of trypsin, chymotrypsin, tissue kallkrein (hK1), and kallikrein 2 (kK2). Zn2+ ions hav e 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 assa ys, which may prove instrumental to uncovering the role of PSA in cancer an d reproduction The inhibition data indicate that Zn2+ could regulate PSA ac tivity, which may prove important in the development of efficient inhibitor s of PSA activity. Prostate 45:132-139, 2000, (C) 2000 Wiley-Liss, Inc.