JBC, Vol. 253, Issue 23, 8593-8597, Dec, 1978
The mechanism of quenching of liver alcohol dehydrogenase fluorescence due to ternary complex formation
W. R. Laws and J. D. Shore
Difference fluorescence emission spectra, reciprocal Stern-Volmer plots,
and variable excitation wave-lengths have been used to evaluate the
selective quenching of the two tryptophan residues/subunit of liver alcohol
dehydrogenase. Trp-15, at the surface of the enzyme, is quenched by KI
consistent with a collisional mechanism, and has a blue-shifted excitation
and red-shifted emission spectrum when compared with the spectral
properties of TRP-314, which is in a hydrophobic milieu at the subunit
interface of the dimeric enzyme. With excitation at 295 nm, Trp-314 is 80%
quenched by formation of a ternary enzyme.NAD+.trifluoroethanol complex,
and the quenching is essentially additive to that caused by KI. Alkaline pH
also results in selective quenching of Trp-314. These results, and
considerations of the three-dimensional structure of the enzyme, indicate
that the quenching of protein fluorescence of liver alcohol dehydrogenase
by either ternary complex formation or alkaline pH is due to resonance
energy transfer to tyrosinate. Likely candidates as energy acceptors are
the Tyr-286 residues are within transfer distance for each Trp-314 residue,
as well as being at the surface of the enzyme and 30 A from the active
center zinc atom. Alkaline pH directly ionizes this tyrosine residue, while
ternary complex formation causes a conformational change resulting in its
ionization.
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