JBC, Vol. 250, Issue 16, 6197-6203, Aug, 1975
Fluorimetric and spectrophotometric studies of DPN-linked isocitrate dehydrogenase from bovine heart. Properties of tyrosyl and tryptophyl residues
C. C. Fan, L. A. Tomcho and G. W. Plaut
The emission maximum of DPN-linked isocitrate dehydrogenase in pH 7.07
buffer is shifted from 317 to 324 nm and fluorescence intensity is
decreased when the excitation wave-length is varied from 270 to 290 nm; in
0.2 M KOH, where the fluorescence of tyrosyl residues is almost completely
quenched, a further substantial decline in quantum yield of protein
fluorescence and a red shift of the emission peak to 339 nm occur. The
latter should be due mainly to tryptophyl residues. The enzyme contains 9.4
tyrosyl residues per subunit of molecular weight 42,000 determined
spectrophotometrically (295 nm) at pH 13, in good agreement with a tyrosine
content of 9.7 by amino acid analysis. No more than 1.1 tyrosyl residues
per subunit can be detected up to pH 10.6 at 7 degrees upon prolonged
incubation. The increase in absorption at 295 nm with increasing pH is
related to loss of enzyme activity and results in a red shift of the
emission maximum, and decreased fluorescence intensity. Treatment of the
enzyme in a Li+-containing buffer at pH 7.5 with an excess of
N-acetylimidazole results in (a) modification of 1.1 tyrosyl residues per
subunit, (b) a 30% decrease in enzyme activity, (c) a 6-nm red shift in
emission maximum, and (d) a decrease in fluorescence intensity. Manganous
DL-isocitrate (1.06 mM) prevents the acetylation of the enzyme.
Deacetylation of the O-acetylated enzyme by hydroxylamine completely
restores the enzyme activity and reverses the spectral changes. The
acetylation studies indicate that the reactive tyrosyl residue does not
participate directly in catalysis but may be involved in maintaining the
proper conformation of the active enzyme center. A net of 1 of the 2
tryptophyl residues per subunit is perturbed immediately by a number of
solvents. This perturbation is not affected by manganous isocitrate,
whereas exposure of tyrosyl residues occurs only with time and is prevented
by the substrate. The perturbation of the tryptophyl residue is accompanied
by a red shift of the fluorescence emission maximum. The more exposed
tryptophyl residue may contribute to the energy transfer from protein to
nucleotides since the quenching of protein fluorescence upon binding of
DPN+, DPNH, or ADP by enzyme results in a blue shift of the emission
maximum. Manganous DL-isocitrate (1.06 mM) quenches protein fluorescence by
16% without a shift in emission peak and does not affect the relative
extent of fluorescence quenching induced by the nucleotides.
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