The cooperative binding of phenylalanine to phenylalanine 4‐monooxygenase studied by ¹H‐NMR paramagnetic relaxation

Abstract

The effect of the paramagnetic high-spin Fe(II1) 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), asrwell as of the temperature (1 8 -40 "C) and the external magnetic field strength (100 -600 MHz). From the frequency dependence of the relaxation rate, an effective correlation time (7,) of 4.2( f0.5) x 10-l o s was calculated for the enzyme-substrate complex, which most likely represents the electron spin relaxation rate (zs) for Fe(II1) ( 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 "C decreased from 2.2(+0.05) x lo3 s-l . M-' i n the enzyme as isolated to 1.2(+0.06) x lo3 s-l . M-' in the presence of saturating concentrations of the substrate. The activation energy of the relaxation rate also decreased from 11.3 k 0.8 kJ/mol to -1.5 k 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(II1) 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(II1).Phenylalanine 4-monooxygenase (phenylalanine hydroxylase) catalyzes the hydroxylation of L-phenylalanine to L-tyrosine in the liver, using (6R)-l-erythro-tetrahydrobiopterin (BH,) as the electron donor (Kaufman and Fisher, 1974

Structure of Tetrameric Human Phenylalanine Hydroxylase and Its Implications for Phenylketonuria

“…During the past few years the mechanism of activation has been the subject of intense biochemical and spectroscopic studies, which have showed that activation of PheOH by the substrate is highly cooperative and always accompanied by alterations in the tertiary structure, but the nature of the conformational change has not been ascertained yet. Substrate activation has been proposed to involve cooperativity among all four subunits of the tetramer (33,34) and radiation target analysis has shown that activation of PheOH results in modification of the monomer structure and promotes stronger association at the dimer interface (35). These results are further supported by evidence of increase in volume of the tetrameric protein (30), and by a shift of the dimer 7 tetramer equilibrium toward the tetrameric form (31) upon binding of L-phenylalanine.…”

Probing the Role of Crystallographically Defined/Predicted Hinge-bending Regions in the Substrate-induced Global Conformational Transition and Catalytic Activation of Human Phenylalanine Hydroxylase by Single-site Mutagenesis

“…The tetrameric form of mammalian PAH displays homotropic cooperative binding of the substrate L-Phe, as observed by steady-state enzyme kinetics and equilibrium binding studies (2,8,28,29). The cooperativity has so far primarily been interpreted within the framework of the classical concerted two-state model of Monod et al (30), i.e.…”

Structure/Function Relationships in Human Phenylalanine Hydroxylase

“…The activation of rat PAH by L-Phe has been interpreted to result from a cooperative binding of the substrate either to a putative allosteric/regulatory site, which is distinct from the catalytic site (Phillips et al, 1984a;Shiman et al, 1990Shiman et al, , 1994Parniak, 1987), or to the active site near the iron (Martinez et al, 1990(Martinez et al, , 1993. The positive cooperativity of LPhe binding seems to reflect changes in the quaternary structure coupled to changes in the tertiary structure of the subunits, by which a form with low-affinity for L-Phe and low-activity (Tform) is converted to a form with high-affinity and high-activity (R-form) (Kaufman, 1993).…”

“…The activation by L-Phe has been found to result in concomitant conformational changes involving the tertiary and quaternary structure (Phillips et al, 1984b;Kappock et al, 1995), changing the tetramer -dimer equilibrium towards the tetrameric form both for rPAH (D@skeland et al, 1982;Parniak, 1987) and recombinant hPAH (Martinez et al, 1995). Moreover, L-Phe binding seems to induce the displacement of a water molecule which is coordinated to the iron in the resting, non-activated rat enzyme (Martinez et al, 1993).…”

“…The activation of rPAH has been interpreted to result from a cooperative binding of L-Phe either to a putative allosteric regulatory site (Parniak and Kaufman, 1981 ;Phillips et al, 1984a;Shiman et al, 1990Shiman et al, , 1994 or to the active site near the iron (Martinez et al, 1991(Martinez et al, , 1993. The activation by L-Phe has been found to result in concomitant conformational changes involving the tertiary and quaternary structure (Phillips et al, 1984b;Kappock et al, 1995), changing the tetramer -dimer equilibrium towards the tetrameric form both for rPAH (D@skeland et al, 1982;Parniak, 1987) and recombinant hPAH (Martinez et al, 1995).…”