Hydrogen Tunneling in Peptidylglycine α-Hydroxylating Monooxygenase

Francisco, Wilson A.; Knapp, Michael J.; Blackburn, Ninian J.; Klinman, Judith P.

doi:10.1021/ja025758s

Cited by 129 publications

(177 citation statements)

References 28 publications

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“…Several recent gas-phase calculations for a small model reaction also resulted in a Swain-Schaad exponent that did not deviate significantly from its semi-classical value at ambient temperature (300-350 K; Kiefer & Hynes 2003Tautermann et al 2004;. Furthermore, even if the magnitude of the exponent (3.3) was altered for a full tunnelling model, it is not expected to change over the narrow temperature range under study and, hence, should not affect the trend manifested in the temperature dependence of the intrinsic isotope effects (Francisco et al 2002;Sikorski et al 2004). To eliminate other common assumptions associated with this procedure (Northrop 1991), the overall reaction needs to be irreversible.…”

Section: Methodsmentioning

confidence: 89%

“…Values of intrinsic H/T, D/T and (H/D) hydrogen/deuterium KIEs were calculated numerically using the appropriate modification of equation (2.3) (Northrop 1991;Cleland 2005). Standard errors on the intrinsic KIEs were estimated by calculating the intrinsic KIEs from the raw data (without an averaging procedure) followed by standard error analysis as described by Francisco et al (2002). The isotope effects on the activation parameters for the intrinsic KIEs were calculated by fitting them into the Arrhenius equation for KIEs: Miller & Benkovic 1998a,b).…”

Section: Methodsmentioning

confidence: 99%

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The role of enzyme dynamics and tunnelling in catalysing hydride transfer: studies of distal mutants of dihydrofolate reductase

Wang

Goodey

Benkovic

et al. 2006

Phil. Trans. R. Soc. B

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Residues M42 and G121 of Escherichia coli dihydrofolate reductase (ecDHFR) are on opposite sides of the catalytic centre (15 and 19 Å away from it, respectively). Theoretical studies have suggested that these distal residues might be part of a dynamics network coupled to the reaction catalysed at the active site. The ecDHFR mutant G121V has been extensively studied and appeared to have a significant effect on rate, but only a mild effect on the nature of H-transfer. The present work examines the effect of M42W on the physical nature of the catalysed hydride transfer step. Intrinsic kinetic isotope effects (KIEs), their temperature dependence and activation parameters were studied. The findings presented here are in accordance with the environmentally coupled hydrogen tunnelling. In contrast to the wild-type (WT), fluctuations of the donor-acceptor distance were required, leading to a significant temperature dependence of KIEs and deflated intercepts. A comparison of M42W and G121V to the WT enzyme revealed that the reduced rates, the inflated primary KIEs and their temperature dependences resulted from an imperfect potential surface prearrangement relative to the WT enzyme. Apparently, the coupling of the enzyme's dynamics to the reaction coordinate was altered by the mutation, supporting the models in which dynamics of the whole protein is coupled to its catalysed chemistry.

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Section: Methodsmentioning

confidence: 89%

Section: Methodsmentioning

confidence: 99%

The role of enzyme dynamics and tunnelling in catalysing hydride transfer: studies of distal mutants of dihydrofolate reductase

Wang

Goodey

Benkovic

et al. 2006

Phil. Trans. R. Soc. B

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“…The detection of sizeable O-18 isotope effects on k cat /K m (O 2 ), which are perturbed by substrate deuteration (21), means that any O 2 chemistry that occurs before loss of hydrogen from substrate must be a fully reversible process (17). The actual transfer of hydrogen occurs by a tunneling mechanism (22), indicating that the origin of the O-18 isotope effect lies with any preequilibrium reduction of O 2 together with heavy atom motions within O 2 that are necessary for effective tunneling of hydrogen from reactant to the O 2 acceptor. Finally, there are no active site residues other than copper and its ligands, which appear to play a direct role in bond cleavage (13, 14, 17).…”

Section: Resultsmentioning

confidence: 99%

“…However, a detailed analysis of the effects of substrate structure and deuteration on O-18 isotope effects (21) was found to be inconsistent with the earlier proposed Cu(II)-OOH 2 and suggested a reductive cleavage of this intermediate to generate copper-oxo as the hydroxylating agent (IV in Scheme 2). This interpretation assumed classical behavior of hydrogen during transfer from substrate to oxygen, which has now been shown in the case of PHM to be dominated by hydrogen tunneling (22). Additionally, recent site-specific mutagenesis studies with PHM unambiguously eliminates a role for the most plausible active site candidate (PHM: Y318; D␤M: Y484) in reductive activation of Cu(II)-OOH (17).…”

mentioning

confidence: 95%

Evidence That Dioxygen and Substrate Activation Are Tightly Coupled in Dopamine β-Monooxygenase

Evans

Ahn

Klinman

2003

Journal of Biological Chemistry

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177

268

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Oxygen activation occurs at a wide variety of enzyme active sites. Mechanisms previously proposed for the copper monooxygenase, dopamine ␤-monooxygenase (D␤M), involve the accumulation of an activated oxygen intermediate with the properties of a copper-peroxo or copper-oxo species before substrate activation. These are reminiscent of the mechanism of cytochrome P-450, where a heme iron stabilizes the activated O 2 species. Herein, we report two experimental probes of the activated oxygen species in D␤M. First, we have synthesized the substrate analog, ␤,␤-difluorophenethylamine, and examined its capacity to induce reoxidation of the prereduced copper sites of D␤M upon mixing with O 2 under rapid freeze-quench conditions. This experiment fails to give rise to an EPR-detectable copper species, in contrast to a substrate with a C-H active bond. This indicates either that the reoxidation of the enzyme-bound copper sites in the presence of O 2 is tightly linked to C-H activation or that a diamagnetic species Cu(II)-O 2 ⅐ ⅐ has been formed. In the context of the open and fully solvent-accessible active site for the homologous peptidylglycine-␣-hydroxylating monooxygenase and by analogy to cytochrome P-450, the accumulation of a reduced and activated oxygen species in D␤M before C-H cleavage would be expected to give some uncoupling of oxygen and substrate consumption. We have, therefore, examined the degree to which O 2 and substrate consumption are coupled in D␤M using both end point and initial rate experimental protocols. With substrates that differ by more than three orders of magnitude in rate, we fail to detect any uncoupling of O 2 uptake from product formation. We conclude that there is no accumulation of an activated form of O 2 before C-H abstraction in the D␤M and peptidylglycine-␣-hydroxylating monooxygenase class of copper monooxygenases, presenting a mechanism in which a diamagnetic Cu(II)-superoxo complex, formed initially at very low levels, abstracts a hydrogen atom from substrate to generate Cu(II)-hydroperoxo and substrate-free radical as intermediates. Subsequent participation of the second copper site per subunit completes the reaction cycle, generating hydroxylated product and water. Dopamine ␤-monooxygenase (D␤M) 1 along with peptidylglycine-␣-hydroxylating monooxygenase (PHM) comprise a unique class of enzymes that contain only copper as a cofactor and catalyze the cleavage of O 2 to form hydroxylated product and water. D␤M is of central importance in the catecholamine biosynthetic pathway, catalyzing the conversion of dopamine to norepinephrine (Scheme 1, top), where both substrate and product serve as neurotransmitters within the central nervous system (1). Primarily localized within the secretory granules of adrenal chromaffin cells and neurons, D␤M is a large, tetrameric glycoprotein (75 kDa per monomer) consisting of two disulfide-linked dimers.Although no crystal structure has been reported, extensive structural data exist for D␤M. Extended X-ray absorption fine structure was used to char...

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“…As observed initially in the reaction catalysed by soybean lipoxygenase (SLO; Glickman et al 1994;Hwang & Grissom 1994) and, subsequently, in a wide range of other enzyme-catalysed C-H abstraction reactions (e.g. Basran et al 1999Basran et al , 2001Kohen et al 1999;Abad et al 2000;Harris et al 2000;Francisco et al 2002;Sikorski et al 2004), the value of A 1 /A 2 often lies considerably above unity. In one case (dihydrofolate reductase, (Sikorski et al 2004)), a complex set of conditions have been postulated to explain A 1 /A 2 [1 within the tenets of variational transition state theory (VTST; Pu et al 2005).…”

Section: Temperature Dependence Of Isotope Effects As a Measure Of Hymentioning

confidence: 89%

Linking protein structure and dynamics to catalysis: the role of hydrogen tunnelling

Klinman

2006

Phil. Trans. R. Soc. B

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Early studies of enzyme-catalysed hydride transfer reactions indicated kinetic anomalies that were initially interpreted in the context of a 'tunnelling correction'. An alternate model for tunnelling emerged following studies of the hydrogen atom transfer catalysed by the enzyme soybean lipoxygenase. This invokes full tunnelling of all isotopes of hydrogen, with reaction barriers reflecting the heavy atom, environmental reorganization terms. Using the latter approach, we offer an integration of the aggregate data implicating hydrogen tunnelling in enzymes (i.e. deviations from Swain-Schaad relationships and the semi-classical temperature dependence of the hydrogen isotope effect). The impact of site-specific mutations of enzymes plays a critical role in our understanding of the factors that control tunnelling in enzyme reactions.

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Hydrogen Tunneling in Peptidylglycine α-Hydroxylating Monooxygenase

Cited by 129 publications

References 28 publications

The role of enzyme dynamics and tunnelling in catalysing hydride transfer: studies of distal mutants of dihydrofolate reductase

The role of enzyme dynamics and tunnelling in catalysing hydride transfer: studies of distal mutants of dihydrofolate reductase

Evidence That Dioxygen and Substrate Activation Are Tightly Coupled in Dopamine β-Monooxygenase

Linking protein structure and dynamics to catalysis: the role of hydrogen tunnelling

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