Thermostability of alcohol dehydrogenase: Evidence for distinct subunits with different deactivation properties

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Abstract
Several independent experimental techniques, including nondenaturing and denaturing isoelectric focusing, spin labeling, and enzyme immobilization, indicate that four ethanol‐active subunits of horse liver alcohol dehydrogenase (LADH) can be classified as one of two types, designated E1 and E2. Thermal inactivation studies of LADH in solution and immobilized to two different supports demonstrate that the first‐order rate constants of deactivation of E1 and E2 differ by more than an order of magnitude. Furthermore, E1, and E2 can be distinguished by EPR spectroscopy, with the less stable subunit type, E2, appearing to have the less compactly structured active‐site environment. The less stable enzyme form also loses catalytic activity upon covalent attachment to CNBr‐Sepharose but remains active when adsorbed to Octyl‐Sepharose. Moreover, the immobilization results in conjunction with lysine modification studies suggest that E2 immobilized to CNBr‐Sepharose cannot bind coenyzme. Overall, these results illustrate how EPR measurements in concert with activity assays can pro vide insights into the molecular mechanisms of enzyme stabilization.