Catalytic properties and active-site structural features of immobilized horse liver alcohol dehydrogenase

Alcohol dehydrogenase from horse liver was immobilized by covalent attachment to CNBr-Sepharose and by adsorption to octyl-Sepharose CL-4B, a hydrophobic analog of Sepharose. In each case, rate constants for the binding and release of coenzyme and for the oxidation of substrates were measured based on the concentration of accessible active-site zinc atoms determined by titration with a paramagnetic inhibitor. All rate constants were substantially reduced upon immobilization; however, the rate constant of immobilized enzyme for ethanol oxidation was independent of the immobilization method, whereas the rate constant for cyclohexanol oxidation was lower for enzyme immobilized to octyl-Sepharose. Consequently, the substrate specificity of the two immobilized enzyme samples differed by an order of magnitude. Moreover, EPR spectroscopy studies and computer graphic analyses of spin labels occupying three defined regions of the active-site domain indicated that the active-site conformation adjacent to the catalytic zinc atom was similar in the two samples while the conformation slightly further from the zinc atom was different. This result may explain why the two immobilized enzyme preparations exhibited the same rate constant toward a small substrate (ethanol) yet different rate constants toward a larger substrate (cyclohexanol), whose rate constant is expected to be sensitive to a larger portion of the active site.