Comparison of inactivation and unfolding of yeast alcohol dehydrogenase during thermal denaturation.

It has been reported that inactivation occurs before noticeable conformational change can be detected during denaturation of creatine kinase (ATP:creatine N-phosphotransferase, EC 2.7.3.2) and other enzymes by guanidinium chloride or urea. It has therefore been suggested that enzyme active sites may display more conformational flexibility than the enzyme molecules as a whole. The present paper compares the inactivation and unfolding of yeast alcohol dehydrogenase during thermal denaturation. Under identical conditions, inactivation takes place before noticeable conformational changes. Kinetics of unfolding can be resolved into two phases. For a given temperature, the fast phase rates are about one order of magnitude slower than the inactivation rates of the free enzyme and approximately the same magnitude as the inactivation rates of enzyme-substrate complexes. This is general accord with the suggestion made previously by Tsou, indicating that the active sites of metal enzymes are situated in a region more flexible than the molecules as a whole.