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 beta-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.
Weber
Wolfgang
W
Institut für Biochemie und Molekularbiologie I, Universitätsklinikum Hamburg-Eppendorf, D-20246 Hamburg, Germany. weber@uke.uni-hamburg.de
Buck
Friedrich
F
Meyer
Arne
A
Hilz
Helmuth
H
D000595:Amino Acid Sequence; D003545:Cysteine / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry; D004229:Dithiothreitol / Q000737:chemistry*; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D017430:Prostate-Specific Antigen / Q000737:chemistry*; D011487:Protein Conformation; D020840:Tissue Kallikreins / Q000737:chemistry
D004220:Disulfides; D020840:Tissue Kallikreins; D017430:Prostate-Specific Antigen; D003545:Cysteine; D004229:Dithiothreitol