THE RATE OF THERMAL INACTIVATION OF TORPEDO ACETYLCHOLINESTERASE IS NOT REDUCED IN THE C231S MUTANT

The rate of thermal inactivation of Torpedo AChE at pH 8.5 was increas ed by the sulfhydryl reagent 5,5'-dithiobis-(2-nitrobenzoic acid) (DTN B). At 30 degrees C or 37 degrees C, inactivation rates with 0.3 mM DT NB increased about 5-fold for the wild-type enzyme and for two site-sp ecific mutants, D72S and V129R. The reversible active site inhibitor, ambenonium, completely stabilized the wild type enzyme and partially s tabilized the D72S mutant. However, ambenonium did not protect against the destabilization introduced by DTNB, which still accelerated inact ivation of D72S 5-fold. When the only free sulfhydryl group in AChE wa s removed by replacing cysteine 231 with serine, increased rates of th ermal inactivation were observed. The inactivation rate incrased by a factor of 2 to 3 for the single mutant (C231S) and by a factor of 5 fo r the double mutant V129R/C231S. Even in the C231S mutants, DTNB still had an additional effect. It increased the inactivation rate for C231 S and V129R/C231 by a factor of about 1.5 to 3 beyond the rates seen i n 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.