Nanosecond pulse fluorometry of conformational change in phenylalanine hydroxylase associated with activation

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Abstract
Conformational change in rat liver phenylanine hydroxylanine hydroxylase associated with activation by phenylalanine or N-(1-anilinipaphth-4-yl)maleimide was investigated by measuring fluorescence specta and flourescence lifetimes of tryptophanyl residues as well as the probe fluorophore conjugated with SH groups of the hydroxylase. The flourescence spectrum of tryptophan exhibited its maximum at 342 nm. It shifted by 8 nm toward longer wavelength accompanied by an increase in its intensity, by preincubation with 1 mM phenylalanine. The fluorescence intensity of tryptophan increased by 36% upon the activation. On the other hand, the binding of (6R)-L-erythro-tetahydrobiopterin, a natural confactor of the enzyme, induced a decrease inthe flourescence intensity by 79% without a shift of the maximum wavelength. The fluorescence lifetime of tryptophan of phenylalanine hydroxylase exhibited two components with lifetimes of 1.7 and 4.1 ns. The values of the lifetimes changed to 1.4 and 5.6 ns, respectively, upon the activation. It is considered that the change in the longer lifetime is correlated with the shift of the emission peak upon the activiation. The values of both the lifetimes decreased to 0.64 and 3.6 ns upon the binding of (6R)-L-erythro-tetrahydrobiopterin, which is coincident with the decrease in the fluorescence intensity. Conjugation of N-(1-anilinonaphth-4-yl)maleimide with SH of phenylalanine hydroxylase brought about a decrease in both the fluorescence intensity and the value of the shorter lifetime ofthe tryptophanyl residues, while the longer lifetime remained unchanged. These changes could be ascribed to excitation energy transfer from tryptophan with the shorter lifetime to the anilinonaphthyl group. When N-(1-anilinonaphth-4-yl)maleimide-conjugated phenylalanine hydroxylase was incubated with 1 mM phenylalanine, the energy-transfer efficiency decreased. The distances between both the tryptophan residues and the probe molecule are considered to be not very short compared to the critical transfer distance (1.7-1.8 nm). The fluorescence lifetime of N-(1-anilinonaphth-4-yl)maleimide exhibited a single component of 4.6 ns. This suggests that the surroundings of the anilinonaphthyl group are homogeneous in phenylalanine hydroxylase. It became heterogeneous upon activation by preincubation with 1 mM phenylalanine or binding of (6R)-L-erythrotetrahydrobiopterin, so that a hydrophobic environment appeared around the fluorophore.