J. Biol. Chem., Vol. 258, Issue 20, 12184-12189, Oct, 1983
The effect of cryosolvents on the spectral and catalytic properties of liver alcohol dehydrogenase
MA Geeves, SC Koerber, MF Dunn and AL Fink
The effects of aqueous organic cryosolvents on the structural and catalytic
properties of horse liver alcohol dehydrogenase (liver alcohol
dehydrogenase) have been investigated. The cosolvents studied were ethanol,
methanol, dimethyl sulfoxide, and dimethylformamide. All show potential as
cosolvents for cryoenzymological investigations of the catalytic action of
liver alcohol dehydrogenase. Limitations due to the formation of abortive
complexes, or the cosolvent acting as a substrate were considered. Possible
adverse structural effects of the cosolvents were ascertained by utilizing
the intrinsic fluorescent properties of the enzyme. Catalytic effects, as
inferred from steady state kinetic studies, were determined from both the
oxidation of ethanol and the reduction of p-nitroso-N,N-dimethylaniline, a
chromophoric aldehyde analog. It is concluded that each of these solvent
systems may be useful for studying certain aspects of the liver alcohol
dehydrogenase catalytic mechanism at subzero temperatures. Thus, although
the formation of ternary enzyme-cosolvent complexes may restrict the use of
cosolvents in some experiments, no apparent adverse effects are observed on
the enzyme structure, coenzyme binding, or catalytic reactions. A number of
interesting features were observed. For example, fluorescence titration of
the native enzyme near 0 degrees C in either aqueous solution or 50%
dimethyl sulfoxide revealed pK values in the vicinity of 10.5 and 12.5, in
contrast to the previously reported single pK of 9.2 observed in aqueous
solution at 25 degrees C.
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