TY - JOUR
T1 - The rate of thermal inactivation of Torpedo acetylcholinesterase is not reduced in the C231S mutant
AU - Wilson, Erica J.
AU - Massoulié, Jean
AU - Bon, Suzanne
AU - Rosenberry, Terrone L.
N1 - Funding Information:
Acknowledgements: This work was supported by a stipend from the Summer Research Program of the NIH Heart, Lung and Blood Institute (to EJW), by NIH grant NS16577, and by grants from mthe Muscular Dystrophy Association of America; Centre National de la Rrecherche Scientifique (CNRS); Direction de la Recherche et de la Technologie (DRET); Association Francaise contre les Myopathies (AFM); and the Human Capital and Mobility program of the European Communities.
PY - 1996/1/29
Y1 - 1996/1/29
N2 - The rate of thermal inactivation of Torpedo AChE at pH 8.5 was increased by the sulfhydryl reagent 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), At 30°C or 37°C, inactivation rates with 0.3 mM: DTNB increased about 5-fold for the wild-type enzyme and for two site-specific mutants, D72S and V129R. The reversible active: site inhibitor, ambenonium, completely stabilized the wild type enzyme and partially stabilized the D72S mutant. However, ambenonium did not protect against the destabilization introduced by DTNB, which still accelerated inactivation of D72S 5-fold. When the only free sulfhydryl group in AChE was removed by replacing cysteine 231 with serine, increased rates of thermal inactivation were observed. The inactivation rate increased by a factor of 2 to 3 for the single mutant (C231S) and by a factor of 5 for the double mutant V129R/C231S. Even in the C231S mutants, DTNB still had an additional effect. It increased the inactivation rate for C231S and V129R/C231 by a factor of about 1.5 to 3 beyond the rates seen in the absence of DTNB. Therefore, at least part of the destabilization seen with DTNB in enzymes that retain C231 does not involve reaction of DTNB with C231.
AB - The rate of thermal inactivation of Torpedo AChE at pH 8.5 was increased by the sulfhydryl reagent 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), At 30°C or 37°C, inactivation rates with 0.3 mM: DTNB increased about 5-fold for the wild-type enzyme and for two site-specific mutants, D72S and V129R. The reversible active: site inhibitor, ambenonium, completely stabilized the wild type enzyme and partially stabilized the D72S mutant. However, ambenonium did not protect against the destabilization introduced by DTNB, which still accelerated inactivation of D72S 5-fold. When the only free sulfhydryl group in AChE was removed by replacing cysteine 231 with serine, increased rates of thermal inactivation were observed. The inactivation rate increased by a factor of 2 to 3 for the single mutant (C231S) and by a factor of 5 for the double mutant V129R/C231S. Even in the C231S mutants, DTNB still had an additional effect. It increased the inactivation rate for C231S and V129R/C231 by a factor of about 1.5 to 3 beyond the rates seen in the absence of DTNB. Therefore, at least part of the destabilization seen with DTNB in enzymes that retain C231 does not involve reaction of DTNB with C231.
KW - Acetylcholinesterase
KW - Disulfide reagent
KW - Site-specific mutagenesis
KW - Thermal inactivation
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U2 - 10.1016/0014-5793(95)01504-3
DO - 10.1016/0014-5793(95)01504-3
M3 - Article
C2 - 8635584
AN - SCOPUS:0030043827
SN - 0014-5793
VL - 379
SP - 161
EP - 164
JO - FEBS Letters
JF - FEBS Letters
IS - 2
ER -