pH-dependent conformational states of horse liver alcohol dehydrogenase.

ABSTRACT

The quenching of liver alcohol dehydrogenase protein fluorescence at alkaline pH indicates two conformational states of the enzyme with a pKa of 9.8+/-0.2, shifted to 10.6+/-0.2 in D2O. NAD+ and 2-p-toluidinonaphthalene-6-sulfonate, a fluorescent probe competitive with coenzyme, bind to the acid conformation of the enzyme. The pKa of the protein-fluorescence quenching curve is shifted toward 7.6 in the presence of NAD+, and the ternary complex formation with NAD+ and trifluoroethanol results in a pH-independent maximal quench. At pH (pD) 10.5, the rate constant for NAD+ binding was 2.6 times faster in D2O2 than in H2O due to the shift of the pKa. Based on these results, a scheme has been proposed in which the state of protonation of an enzyme functional group with a pKa of 9.8 controls the conformational state of the enzyme. NAD+ binds to the acid conformation and subsequently causes another conformational change resulting in the perturbation of the pKa to 7.6. Alcohol then binds to the unprotonated form of the functional group with a pKa of 7.6 in the binary enzyme-NAD+ complex and converts the enzyme to the alkaline conformation. Thus, at neutral pH liver alcohol dehydrogenase undergoes two conformational changes en route to the ternary complex in which hydride transfer occurs.