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

TY - JOUR

T1 - Prostate specific antigen: one out of five disulfide bridges determines inactivation by reduction.

AU - Weber, Wolfgang

AU - Buck, Friedrich

AU - Meyer, Arne

AU - Hilz, Helmuth

PY - 2009

Y1 - 2009

N2 - 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.

AB - 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.

M3 - SCORING: Zeitschriftenaufsatz

VL - 379

SP - 1101

EP - 1106

JO - BIOCHEM BIOPH RES CO

JF - BIOCHEM BIOPH RES CO

SN - 0006-291X

IS - 4

M1 - 4

ER -