JBC, Vol. 252, Issue 2, 433-436, Jan, 1977
pH-dependent conformational states of horse liver alcohol dehydrogenase
J. K. Wolfe, C. F. Weidig, H. R. Halvorson and J. D. Shore
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.
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