REVERSIBLE CONFORMATIONAL TRANSITION GIVES RISE TO ZIGZAG TEMPERATURE-DEPENDENCE OF THE RATE-CONSTANT OF IRREVERSIBLE THERMOINACTIVATION OFENZYMES

We have obtained unusual 'zig-zag' temperature dependencies of the rat e constant of irreversible thermoinactivation (k(in)) of enzymes (alph a-chymotrypsin, covalently modified alpha-chymotrypsin, and ribonuclea se) in a plot of log k(in) versus reciprocal temperature (Arrhenius pl ot). These dependencies are characterized by the presence of both asce nding and descending linear portions which have positive and negative values of the effective activation energy (E(a)), respectively. A kine tic scheme has been suggested that fits best for a description of thes e zig-zag dependencies. A key element of this scheme is the temperatur e-dependent reversible conformational transition of enzyme from the 'l ow-temperature' native state to a 'high-temperature' denatured form; t he latter form is significantly more stable against irreversible therm oinactivation than the native enzyme. A possible explanation for a dif ference in thermal stabilities is that low-temperature and high-temper ature forms are inactivated according to different mechanisms. Existen ce of the suggested conformational transition was proved by the method s of fluorescence spectroscopy and differential scanning calorimetry. The values of Delta H and Delta S for this transition, determined from calorimetric experiments, are highly positive; this fact underlies a conclusion that this heat-induced transition is caused by an unfolding of the protein molecule. Surprisingly, in the unfolded high-temperatu re conformation, alpha-chymotrypsin has a pronounced proteolytic activ ity, although this activity is much smaller than that of the native en zyme.