THE DENATURATION AND DEGRADATION OF STABLE ENZYMES AT HIGH-TEMPERATURES

Now that enzymes are available that are stable above 100 degrees C it is possible to investigate conformational stability at this temperatur e, and also the effect of high-temperature degradative reactions in fu nctioning enzymes and the inter-relationship between degradation and d enaturation. The conformational stability of proteins depends upon sta bilizing forces arising from a large number of weak interactions, whic h are opposed by an almost equally large destabilizing force due mostl y to conformational entropy. The difference between these, the net fre e energy of stabilization, is relatively small, equivalent to a few in teractions. The enhanced stability of very stable proteins can be achi eved by an additional stabilizing force which is again equivalent to o nly a few stabilizing interactions. There is currently no strong evide nce that any particular interaction (e.g. hydrogen bonds, hydrophobic interactions) plays a more important role in proteins that are stable at 100 degrees C than in those stable at 50 degrees C, or that the str uctures of very stable proteins are systematically different from thos e of less stable proteins. The major degradative mechanisms are deamid ation of asparagine and glutamine, and succinamide formation at aspart ate and glutamate leading to peptide bond hydrolysis. In addition to b eing temperature-dependent, these reactions are strongly dependent upo n the conformational freedom of the susceptible amino acid residues. E vidence is accumulating which suggests that even at 100 degrees C deam idation and succinamide formation proceed slowly or not at all in conf ormationally intact (native) enzymes. Whether this is the case at high er temperatures is not yet clear, so it is not known whether denaturat ion or degradation will set the upper limit of stability for enzymes.