Browsing by Subject "Tyrosine Hydroxylase"
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Item Spectroscopic and Kinetic Investigation of the Catalytic Mechanism of Tyrosine Hydroxylase(2011-02-22) Eser, Bekir EnginTyrosine Hydroxylase (TyrH) is a pterin-dependent mononuclear non-heme iron oxygenase. TyrH catalyzes the hydroxylation reaction of tyrosine to dihydroxyphenylalanine (DOPA). This reaction is the first and the rate-limiting step in the biosynthesis of the catecholamine neurotransmitters. The active site iron in TyrH is coordinated by the common facial triad motif, 2-His-1-Glu. A combination of kinetic and spectroscopic techniques was applied in order to obtain insight into the catalytic mechanism of this physiologically important enzyme. Analysis of the TyrH reaction by rapid freeze-quench Mossbauer spectroscopy allowed the first direct characterization of an Fe(IV) intermediate in a mononuclear nonheme enzyme catalyzing aromatic hydroxylation. Further rapid kinetic studies established the kinetic competency of this intermediate to be the long-postulated hydroxylating species, Fe(IV)O. Spectroscopic investigations of wild-type (WT) and mutant TyrH complexes using magnetic circular dichroism (MCD) and X-ray absorption spectroscopy (XAS) showed that the active site iron is 6-coordinate in the resting form of the enzyme and that binding of either tyrosine or 6MPH4 alone does not change the coordination. However, when both tyrosine and 6MPH4 are bound, the active site becomes 5-coordinate, creating an open site for reaction with O2. Investigation of the kinetics of oxygen reactivity of TyrH complexes in the absence and presence of tyrosine and/or 6MPH4 indicated that there is a significant enhancement in reactivity in the 5-coordinate complex in comparison to the 6-coordinate form. Similar investigations with E332A TyrH showed that Glu332 residue plays a role in directing the protonation of the bridged complex that forms prior to the formation of Fe(IV)O. Rapid chemical quench analyses of DOPA formation showed a burst of product formation, suggesting a slow product release step. Steady-state viscosity experiments established a diffusional step as being significantly rate-limiting. Further studies with stopped-flow spectroscopy indicated that the rate of TyrH reaction is determined by a combination of a number of physical and chemical steps. Investigation of the NO complexes of TyrH by means of optical absorption, electron paramagnetic resonance (EPR) and electron spin echo envelope modulation (ESEEM) techniques revealed the relative positions of the substrate and cofactor with respect to NO, an O2 mimic, and provided further insight into how the active site is tuned for catalytic reactivity upon substrate and cofactor binding.Item Studies of the chemical and regulatory mechanisms of tyrosine hydroxylase(Texas A&M University, 2006-08-16) Frantom, Patrick AllenTyrosine hydroxylase (TyrH) catalyzes the pterin-dependent hydroxylation of tyrosine to form dihydroxyphenylalanine. The enzyme requires one atom of ferrous iron for activity. Using deuterated 4-methylphenylalanine substrates, intrinsic primary and secondary isotope effects of 9.6 ?? 0.9 and 1.21 ?? 0.08 have been determined for benzylic hydroxylation catalyzed by TyrH. The large, normal secondary isotope effect is consistent with a mechanism involving hydrogen atom abstraction to generate a radical intermediate. The similarity of the isotope effects to those measured for benzylic hydroxylation catalyzed by cytochrome P-450 suggests that a high-valent, ferryl-oxo species is the hydroxylating species in TyrH. Uncoupled mutant forms of TyrH have been utilized to unmask isotope effects on steps in the aromatic hydroxylation pathway which also implicate a ferryl-oxo intermediate. Inverse secondary isotope effects were seen when 3,5-2H2-tyrosine was used as a substrate for several mutant enzyme forms. This result is consistent with a direct attack by a ferryl-oxo species on the aromatic ring of tyrosine forming a cationic intermediate. Rapid-freeze quench M??ssbauer studies have provided preliminary spectroscopic evidence for an Fe(IV) intermediate in the reaction catalyzed by TyrH. The role of the iron atom in the regulatory mechanism has also been investigated. The iron atom in TyrH, as isolated, is in the ferric form and must be reduced for activity. The iron can be reduced by a number of one-electron reductants including tetrahydrobiopterin, ascorbate, and glutathione; however, it appears that BH4 (kred = 2.8 ?? 0.1 mM-1 s-1) is the most likely candidate for reducing the enzyme in vivo. A one-electron transfer would require a pterin radical. Rapid-freeze quench EPR experiments aimed at detecting the intermediate were unsuccessful, suggesting that it decays very rapidly by reducing another equivalent of enzyme. The active Fe(II) form can also become oxidized by oxygen (210 ?? 30 M-1 s-1); this increases the affinity of catecholamine inhibitors. Serine 40 can be phosphorylated to relieve the inhibition; however, results with S40E TyrH show phosphorylation does not have an effect on the rate constant for reduction of the enzyme but causes a 40% decrease in the rate constant of oxidation.