Nanosecond pulse fluorometry of conformational change in phenylalanine hydroxylase associated with activation.
Conformational change in rat liver phenylalanine hydroxylase associated with activation by phenylalanine or N-(1-anilinonaphth-4-yl)maleimide was investigated by measuring fluorescence spectra and fluorescence lifetimes of tryptophanyl residues as well as the probe fluorophore conjugated with SH groups of the hydroxylase. The fluorescence 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-tetrahydrobiopterin, a natural cofactor of the enzyme, induced a decrease in the fluorescence 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 activation. 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 of the tryptophanyl residues, while the longer lifetime remained unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)
Koizumi
S
S
National Institute of Child Health and Human Development, Bethesda, Maryland 20892.
Tanaka
F
F
Kaneda
N
N
Kano
K
K
Nagatsu
T
T
D000818:Animals; D004789:Enzyme Activation; D007202:Indicators and Reagents; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008301:Maleimides / Q000494:pharmacology; D010649:Phenylalanine / Q000494:pharmacology; D010651:Phenylalanine Hydroxylase / Q000378:metabolism*; D011487:Protein Conformation; D051381:Rats; D013050:Spectrometry, Fluorescence / Q000379:methods; D013997:Time Factors; D014364:Tryptophan
D007202:Indicators and Reagents; D008301:Maleimides; D010649:Phenylalanine; C010854:N-(1-anilinonaphthalyl)-4-maleimide; D014364:Tryptophan; D010651:Phenylalanine Hydroxylase