The characterization of prostate-specific antigen: Identification of the zinc(2+) inhibition site, an activation site and a rationale for the complex regulation of protease activity

Prostate specific antigen (PSA, also known as human kallikrein 3) is an important diagnostic indicator of benign prostatic hyperplasia and prostate cancer. However, the role of PSA in these diseases has not been established. PSA is a serine protease produced by the epithelial cells of the prostate, although its activity is low. The proteolytic activity of PSA is regulated by both salt activation and Zn2+ inhibition. The characterization of PSA was pursued in an effort to correlate these atypical biochemical properties with biological function.; We have discovered that salt enhances PSA activity 103–10 4 fold and citrate is the most efficient salt for in vitro activation. Citrate is believed to be the physiologically relevant activator since it is also a major component of semen and is present in a concentration of up to 0.45 N. The high concentration of citrate required for activation could result from either a weak binding site or an effect on protein conformation resulting from preferential exclusion of salt from the protein surface. To address this issue, I examined arginine residues since they are common constituents of citrate binding sites in other proteins and may represent citrate binding site(s) in PSA. Chemical modification of arginine residues shows that specific binding of citrate exists while activation by the less potent chloride was not similarly affected. Two citrate binding site candidates were probed by mutating two arginine residues in each site. However, neither mutant was less sensitive to citrate activation. The activity and activation of R95_gQ/R95_jQ was the same as wt PSA. However the R36A/R39A mutant confers a tenfold increase in salt independent activity. The mechanism of this enhancement has not been established. Activation of PSA appears to operate via both specific binding and general solvent effect mechanisms.; Zn2+ inhibits PSA with a K_i of 12 μM. The concentration of Zn2+ in prostatic fluid is 7 mM indicating that it is a significant physiological inhibitor. I subjected two prospective Zn2+ sites to mutagenesis. Zn2+ also inhibits H91A PSA with a K_i of 12 μM. However, Zn2+ inhibits H71A/H75A PSA with a K_i of 260 μM. The H71A/H75A mutation disrupts Zn2+ binding demonstrating that one or both of these residues are responsible for Zn2+ inhibition.; The regulation of PSA proteolytic activity is unusually complex. PSA is also found in breast tissue, placenta and in the blood of pregnant women. These observations suggest that PSA may have an additional biological activity. Previous work has shown that PSA is a trophin in cell culture experiments and that prostate cancer cell growth is also stimulated by neurotrophins via Trk receptors. (Abstract shortened by UMI.)...