Prostate specific antigen: One out of five disulfide bridges determines inactivation by reduction

PSA (kallikrein hK3) proteolytic activity proved highly sensitive to reducing agents like dithiothreitol (DTT) and dihydrolipoic acid while β-mercaptoethanol and glutathione were less effective. Ascorbate exhibited no significant inhibitory potential. Loss of activity by reduction could be readily reversed by re-oxidation. Inactivation was associated with the reduction of two out of five conserved disulfides. Mass spectrometry of differentially modified cysteines, and Edman degradation analyses identified Cys 22–Cys 157 and Cys 191–Cys 220 as DDT-sensitive. The highly homologous porcine pancreatic kallikrein (pK1) showed a completely different response: Even at 20 mM DDT, no inactivation was seen; and in this case, only one of the five disulfides (Cys 22–Cys 157) was opened. This indicated that it is the accessabilty of the Cys 191–Cys 220 disulfide near the catalytic serine 195 that decides on the ability of reductants to inactivate the proteolytic activity of PSA. A structural basis for this interpretation is provided when the two homologous proteins were compared with respect to the threedimensional architecture around the crucial disulfide Cys 191–Cys 220 where in the case of PK1, but not in PSA, the phenylalanine-residue (Phe 149) is in an interfering position.