Oxygen-18 Kinetic Isotope Effect Studies of the Tyrosine Hydroxylase Reaction:  Evidence of Rate Limiting Oxygen Activation

Francisco, Wilson A.; Tian, Gaochao; Fitzpatrick, Paul F.; Klinman, Judith P.

doi:10.1021/ja973543q

Cited by 78 publications

(100 citation statements)

References 29 publications

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“…With this enzyme, the rate-limiting step in turnover appears to be the formation of the hydroxylating intermediate since the k cat value is relatively insensitive to the identity of the amino acid substrate (69). There is an 18 O isotope effect on the V/K value for oxygen of 1.0175 (70). This establishes that there is a change in the bond order to oxygen in the rate-determining step.…”

Section: Mechanism Of Oxygen Activationmentioning

confidence: 91%

Mechanism of Aromatic Amino Acid Hydroxylation

2003

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Section: Mechanism Of Oxygen Activationmentioning

confidence: 91%

Mechanism of Aromatic Amino Acid Hydroxylation

2003

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“…Oxygen isotope effects have been examined recently for a series of dioxygen-binding proteins and dioxygen-activating enzymes (12)(13)(14)(15)(16)(17)(18). In these experiments, the proteins or enzymes discriminate between isotopologues in the binding or consumption of dioxygen.…”

Section: Steady-state Oxidation Of Ch 3 Cn Catalyzed By Smmo-mentioning

confidence: 99%

“…Experimental procedures were similar to those described previously for other dioxygenactivating enzymes (12,13,16,17). The reactions employed natural abundance 18 O-labeled dioxygen and examined the extent of discrimination between 18 O-and 16 O-containing dioxygen.…”

Section: Steady-state Oxidation Of Ch 3 Cn Catalyzed By Smmo-mentioning

confidence: 99%

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Oxygen Kinetic Isotope Effects in Soluble Methane Monooxygenase

Stahl

Francisco

Merkx

et al. 2001

Journal of Biological Chemistry

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Soluble methane monooxygenase (sMMO) contains a nonheme, carboxylate-bridged diiron site that activates dioxygen in the catalytic oxidation of hydrocarbon substrates. Oxygen kinetic isotope effects (KIEs) have been determined under steady-state conditions for the sMMO-catalyzed oxidation of CH 3 CN, a liquid substrate analog. Kinetic studies of the steady-state sMMO reaction revealed a competition between fully coupled oxygenase activity, which produced glycolonitrile (HOCH 2 CN) and uncoupled oxidase activity that led to water formation. The oxygen KIE was measured independently for both the oxygenase and oxidase reactions, and values of 1.0152 ؎ 0.0007 and 1.0167 ؎ 0.0010 were obtained, respectively. The isotope effects and separate dioxygen binding studies do not support irreversible formation of an enzyme-dioxygen Michaelis complex. Additional mechanistic implications are discussed in the context of previous data obtained from single turnover and steady-state kinetic studies.The soluble methane monooxygenase system (sMMO) 1 found in certain methanotrophic bacteria catalyzes the oxidation of methane to methanol according to Reaction 1 (1, 2).This reaction is the first essential step of a metabolic pathway that allows these bacteria to use methane as their sole source of energy and carbon. The soluble MMO system comprises three protein components: a 251 kDa hydroxylase (MMOH), a reductase (MMOR) that transfers electrons from NADH to MMOH, and a cofactorless regulatory protein (MMOB). Reductive activation of dioxygen and subsequent methane oxidation take place at a carboxylate-bridged diiron cluster in the active site of MMOH. (3,4,6,8). This observation suggests that Q, or a subsequent unobserved intermediate, is responsible for methane oxidation.Recent kinetic studies and theoretical calculations have suggested the presence of additional intermediates (6, 9, 10). For example, proton transfer steps have been observed in the formation and decay of H peroxo , a result consistent with a protonated peroxo intermediate in the mechanism (9). Other kinetic studies provide indirect evidence for one or more intermediates preceding the formation of H peroxo . These may include an enzyme-dioxygen Michaelis complex or a superoxo intermediate (6, 9 -11).Kinetic and equilibrium oxygen isotope effects have been determined recently for several proteins and enzymes that activate dioxygen (12)(13)(14)(15)(16)(17)(18). Such experiments directly probe reaction steps involved in the activation of dioxygen and can provide unique mechanistic information that may not be available by other means. In this study, oxygen-18 kinetic isotope effects determined for sMMO from M. capsulatus (Bath) together with steady-state kinetics and dioxygen binding studies yield further insights into dioxygen activation by this enzyme system. EXPERIMENTAL PROCEDURESGeneral Considerations-Growth of M. capsulatus (Bath) cells and subsequent purification of MMOH were carried out as described previously (19,20). Both MMOB and MMOR were obtained from recombi...

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“…The other half of the oxygen molecule is found in the hydroxylated product (L-Tyr). It has been postulated that a Fe lx =0 complex is an intermediate in the reaction mechanism (12). The existence of Fe IN -0 has been verified in model compounds by a combination of Mössbauer and X-ray absorption spectroscopy ( 19,20 turn is believed to be responsible for the hydroxylation of the aromatic amino acid.…”

Section: Introductionmentioning

confidence: 98%

The Reaction Mechanism of Phenylalanine Hydroxylase. – A Question of Coordination

Teigen

Jensen²,

Martı́nez³

2005

Pteridines

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Phenylalanine hydroxylase (PAH) is a non-heme iron and tetrahydrobiopterin-dependent enzyme that catalyzes the hydroxylation of L-phenylalanine to L-tyrosine using dioxygen as additional substrate. The cofactor tetrahydrobiopterin accepts one of the oxygen atoms of dioxygen during catalysis and also seems to be involved in prereduction of the active site iron from the ferric to the activated ferrous form. Structures of the truncated form of PAH in complex with substrate and cofactor are available, but the oxygen binding site and the actual mechanism of electron transfer are uncertain. It is believed that dioxygen binds directly to the metal, where it is activated, and several reaction mechanisms involving end-on binding of 0 2 have been proposed based on both experimental studies and quantum mechanical calculations. However, in this work we aimed to investigate the possibility of side-on binding of dioxygen to the iron. Furthermore, NMR and recent high-resolution crystallographic studies also place the cofactor in closer proximity to the iron, challenging the mechanistic conclusions from earlier crystallographic and computational studies. In this paper we report preliminary results from a density functional theory (DFT) study of the coordination of dioxygen to a structural model of PAH based on a recent crystallographic structure. These results are compared with existing computational and experimental data and their implications for the mechanism of the PAH-reaction are discussed. Particular attention is paid to the binding-mode of dioxygen and the iron-cofactor distance.

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Oxygen-18 Kinetic Isotope Effect Studies of the Tyrosine Hydroxylase Reaction: Evidence of Rate Limiting Oxygen Activation

Cited by 78 publications

References 29 publications

Mechanism of Aromatic Amino Acid Hydroxylation

Mechanism of Aromatic Amino Acid Hydroxylation

Oxygen Kinetic Isotope Effects in Soluble Methane Monooxygenase

The Reaction Mechanism of Phenylalanine Hydroxylase. – A Question of Coordination

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