The cooperative binding of phenylalanine to phenylalanine 4-monooxygenase studied by 1H-NMR paramagnetic relaxation. Changes in water accessibility to the iron at the active site upon substrate binding.
The effect of the paramagnetic high-spin Fe(III) ion in phenylalanine 4-monooxygenase (phenylalanine hydroxylase, EC 1.14.16.1) on the water proton longitudinal relaxation rate has been used to study the environment of the iron center. The relaxation rate was measured as a function of the concentration of enzyme, substrate (phenylalanine), inhibitor (noradrenaline) and activator (lysolecithin), as well as of the temperature (18-40 degrees C) and the external magnetic field strength (100-600 MHz). From the frequency dependence of the relaxation rate, an effective correlation time (tau c) of 4.2(+/- 0.5) x 10(-10) s was calculated for the enzyme-substrate complex, which most likely represents the electron spin relaxation rate (tau s) for Fe(III) (S = 5/2) in this complex. The relaxation rate was proportional to the concentration of enzyme (0.04-1 mM) both in the absence and presence of phenylalanine, but the paramagnetic molar relaxivity at 400 MHz and 22 degrees C decreased from 2.2(+/- 0.05) x 10(3) s-1.M-1 in the enzyme as isolated to 1.2(+/- 0.06) x 10(3) s-1.M-1 in the presence of saturating concentrations of the substrate. The activation energy of the relaxation rate also decreased from 11.3 +/- 0.8 kJ/mol to -1.5 +/- 0.2 kJ/mol upon incubation of the enzyme with 5 mM phenylalanine. The results obtained can be interpreted in terms of a slowly exchanging water molecule coordinated to the catalytic paramagnetic Fe(III) in the native and resting enzyme, and that this water molecule seems to be displaced from coordination on the binding of substrate or inhibitor. Moreover, the effect of increasing concentrations of phenylalanine and noradrenaline on the water proton relaxation rate and on the hydrophobic surface properties of the enzyme indicate that substrate and inhibitor induce a similar cooperative conformational change upon binding at the active site. By contrast, the activator lysolecithin does not seem to affect the interaction of water with the catalytic Fe(III).
Martínez
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A
Department of Biochemistry, University of Bergen, Norway.
Olafsdottir
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S
Flatmark
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T
D000818:Animals; D001665:Binding Sites; D066298:In Vitro Techniques; D007501:Iron / Q000737:chemistry; D008099:Liver / Q000201:enzymology; D009682:Magnetic Resonance Spectroscopy; D009638:Norepinephrine / Q000494:pharmacology; D010649:Phenylalanine / Q000378:metabolism*; D010651:Phenylalanine Hydroxylase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D013696:Temperature; D014867:Water / Q000737:chemistry
D014867:Water; D010649:Phenylalanine; D007501:Iron; D010651:Phenylalanine Hydroxylase; D009638:Norepinephrine