pH dependence of free and immobilized yeast alcohol dehydrogenase kinetics

A study was made of the influence of pH on the reaction between NAD and ethanol, catalyzed by yeast alcohol dehydrogenase, both in free solution and attached to the inner surface of a nylon tube. A new least-squares analysis of the results has been devised; it is simpler to apply and is more realistic than those previously employed. Analysis of the results for the free enzyme indicated that the free enzyme has two active ionizing groups having pK values of about 6.6 and 8.8. These pK values undergo only small changes when the enzyme is bound to NAD and when it is bound to both NAD and ethanol. With the immobilized enzyme and saturating concentrations of ethanol the rates went through a maximum as the pH was varied from 6.5 to 10.0. With saturating concentrations of NAD there was a steady increase in rate, with no falling off at pH 10. Immobilization generally brought about an increase in the pK values. These increases are attributed partly to a residual negative surface charge which attracts the leaving H+ ions. They are also attributed partly to the formation in the reaction of H+ ions, which cause the local pH to be lower than that in the bulk solution. This effect is more important with saturating NAD ions, since the buffer anions will then be less mobile and less able to mediate the movement of protons.