Prostate specific antigen: one out of five disulfide bridges determines inactivation by reduction.
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Prostate specific antigen: one out of five disulfide bridges determines inactivation by reduction. / Weber, Wolfgang; Buck, Friedrich; Meyer, Arne; Hilz, Helmuth.
In: BIOCHEM BIOPH RES CO, Vol. 379, No. 4, 4, 2009, p. 1101-1106.Research output: SCORING: Contribution to journal › SCORING: Journal article › Research › peer-review
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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 -