Effect of thermal and chemical denaturants on Thermoanaerobacter ethanolicus secondary-alcohol dehydrogenase stability and activity

Thermoanaerobacter ethanolicus 39E secondary-alcohol dehydrogenase (2 degre es ADH) was optimally active near 90 degrees C displaying thermostability h alf-lives of 1.2 days, 1.7 h, 19 min, 9.0 min, and 1.3 min at 80 degrees C, 90 degrees C, 92 degrees C, 95 degrees C, and 99 degrees C, respectively. Enzyme activity loss upon heating (90-100 degrees C) was accompanied by pre cipitation, but the soluble enzyme remaining after partial inactivation ret ained complete activity. Enzyme thermoinactivation was modeled by a pseudo- first order rate equation suggesting that the rate determining step was uni molecular with respect to protein and thermoinactivation preceded aggregati on. The apparent 2 degrees ADH melting temperature (T-m) occurred at simila r to 115 degrees C, 20 degrees C higher than the temperature for maximal ac tivity, suggesting that it is completely folded in its active temperature r ange. Thermodynamic calculations indicated that the active folded structure of the 2 degrees ADH is stabilized by a relatively small Gibbs energy (Del ta G(stab)(double dagger) = 110 kJ mol(-1)). 2 degrees ADH catalytic activi ties at 37 degrees C to 75 degrees C, were 2-fold enhanced by guanidine hyd rochloride (GuHCl) concentrations between 120 mM and 190 mM. These results demonstrate the extreme resistance of this thermophilic 2 degrees ADH to th ermal or chemical denaturation; and suggest increased temperature or GuHCl levels seem to enhance protein fixability and activity. Published by Elsevi er Science Inc. All rights reserved.