pH and deuterium kinetic isotope effects studies on the oxidation of choline to betaine-aldehyde catalyzed by choline oxidase

doi:10.1016/S1570-9639(03)00188-2

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

Volume 1650, Issues 1–2, 21 August 2003, Pages 4-9

https://doi.org/10.1016/S1570-9639(03)00188-2 Get rights and content

Abstract

The FAD-dependent choline oxidase catalyzes the four-electron oxidation of choline to glycine-betaine, with betaine-aldehyde as intermediate. The enzyme is capable of accepting either choline or betaine-aldehyde as a substrate, allowing the investigation of the reaction mechanism for both the conversion of choline to betaine-aldehyde and of betaine-aldehyde to glycine-betaine. In the present study, pH and deuterium kinetic isotope effects with [1,2-²H₄]-choline were used to study the mechanism of oxidation of choline to betaine-aldehyde. The V/K and V_max pH-profiles increased to limiting values with increasing pH, suggesting the presence of a catalytic base essential for catalysis at the enzyme active site. From the V/K pH-profile with [1,2-²H₄]-choline, a pK_a of 8.0 was determined for the catalytic base. This pK_a was shifted to 7.5 in the V/K pH-profile with choline, indicating a significant commitment to catalysis with this substrate. In agreement with this conclusion, the ^D(V/K) values decreased from a limiting value of 12.4 below pH 6.5 to a limiting value of 4.1 above pH 9.5. The large ^D(V/K) values at low pH are consistent with carbon–hydrogen bond cleavage of choline being nearly irreversible and fully rate-limiting at low pH. Based on comparison of amino acid sequences and previous structural and mechanistic studies on other members of the GMC oxidoreductase superfamily, the identity of the catalytic base of choline oxidase is proposed.

Introduction

Choline oxidase (E.C. 1.1.3.17) was first described in 1977 by Ikuta et al. [1], who reported the purification and initial characterization of the enzyme from the Gram-positive soil bacterium Arthrobacter globiformis. The enzyme catalyzes the four-electron oxidation of choline to glycine-betaine (N,N,N-trimethylglycine; betaine) via two sequential FAD-dependent reactions in which betaine-aldehyde is formed as an intermediate (Scheme 1). In both reactions, molecular oxygen acts as the primary electron acceptor with production of hydrogen peroxide. Based on amino acid sequence alignment, the enzyme can be grouped in the GMC oxidoreductase superfamily, which comprises glucose oxidase, methanol oxidase, cholesterol oxidase, and choline dehydrogenase [2]. The study of the mechanism of choline oxidase is of interest both from fundamental and applied standpoints. Choline oxidase is one of the three reported flavin-dependent enzymes, along with thiamine oxidase (E.C. 1.1.3.23) and choline dehydrogenase (E.C. 1.1.99.1), that catalyze the oxidation of a substrate alcohol to a carboxylic acid via an aldehyde intermediate [3], [4]. Such a four-electron oxidation of unactivated alcohols has been extensively studied on the NAD⁺-dependent histidinol dehydrogenase (E.C. 1.1.1.23), for which a wealth of kinetic and mechanistic information is available [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17]. In contrast, minimal mechanistic data are available on the flavin-dependent oxidoreductases [3]. From a biotechnological standpoint, the development of biosensors for the detection of choline and choline ester derivatives, such as acetylcholine, in serological samples and foods renders choline oxidase of interest for clinical and industrial uses [18], [19], [20], [21], [22]. Furthermore, the recent findings that many bacterial and plant species accumulate glycine-betaine in response to various stresses, such as high salt, high or low temperature, and water deficiency [23], [24], [25], [26], [27], [28], have prompted considerable interest in research on glycine-betaine biosynthesis, with the goal of genetically engineering increased stress tolerance in economically relevant crop plants [29], [30], [31], [32], [33], [34], [35]. To date, despite a wealth of studies on the biotechnological applications of the enzyme, minimal biochemical and mechanistic studies on choline oxidase have been reported. Consequently, the study of the mechanism of choline oxidase offers the opportunity to expand our studies on the mechanism of carbon–hydrogen bond cleavage of unactivated alcohols catalyzed by flavin-dependent enzymes, and compare the mechanistic properties of the flavin-dependent oxidoreductases that catalyze four-electron oxidations of alcohols with those of the pyridine nucleotide-dependent reductases.

Our group has recently reported the analysis of the steady state kinetic mechanism of choline oxidase, which is consistent with the mechanism of Scheme 2 [36]. Briefly, after formation of the E-FAD_ox-C complex, choline is oxidized to form betaine-aldehyde bound to the reduced enzyme. The resulting E-FAD_red-BA complex reacts with oxygen, yielding the E-FAD_ox-BA species, in which the enzyme-bound betaine-aldehyde then partitions between catalysis to yield glycine-betaine bound to the reduced enzyme and dissociation from the enzyme active site to form the E-FAD_ox species. The E-FAD_red-B species finally reacts with oxygen before the release of glycine-betaine. In the present report, we have expanded our initial steady state studies using pH and deuterium isotope effects as mechanistic probes to investigate further the mechanism of oxidation of choline to betaine-aldehyde catalyzed by choline oxidase.

Section snippets

Materials

Choline chloride was from ICN Pharmaceutical, Inc. [1,2-²H₄]-Choline bromide was from Isotec Inc. Choline oxidase from A. globiformis was from Sigma-Aldrich. Stock solutions of choline oxidase were prepared in 100 mM potassium phosphate, pH 7, and stored at −20 °C. The concentration of choline oxidase was determined spectrophotometrically by measuring the absorbance at 454 nm using an Agilent Technologies diode-array spectrophotometer Model HP 8453, with ε₄₅₄=11,300 M⁻¹ cm⁻¹ [37]. All other

pH dependence of the V/K values for choline and [1,2-²H₄]-choline as substrate

The pH dependence of the kinetic parameters with choline as substrate was determined by measuring initial rates of reaction at different concentrations of choline in air-saturated buffer in the pH range 6 to 10. As shown in Fig. 1, the log (V/K) value pH-profile increased with a unit slope with increasing pH reaching a limiting value above pH 8.0. One group with an apparent pK_a value of 7.5±0.2 must be unprotonated for catalysis. When choline was substituted with [1,2-²H₄]-choline as substrate,

Discussion

The mechanism of cleavage of carbon–hydrogen bonds of primary alcohols catalyzed by flavin-dependent enzymes has received considerable attention over the past four decades. Such a reaction is particularly intriguing due to the high-energy barrier associated with the removal of a hydride equivalent from the substrate α-carbon and the fact that such chemistry is carried out on the underivatized substrate. Among the best understood flavin-dependent enzymes that catalyze the oxidation of primary

Acknowledgements

I thank Dr. Paul F. Fitzpatrick for helpful discussions and the reviewers for their insightful suggestions.

This study was supported in part by Grant PRF #37351-G4 from the American Chemical Society, a Research Initiation Grant and a Quality Improvement Fund from Georgia State University.

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The relevance of gating mechanisms for choline and oxygen access to the active site cavity was elucidated. Besides choline, the enzyme can also oxidize betaine aldehyde as a substrate [5–7]. The enzymology of choline oxidase with betaine aldehyde has been investigated to a much lesser extent.
Choline oxidase catalyzes the four-electron, two-step, flavin-mediated oxidation of choline to glycine betaine. The enzyme is important both for medical and biotechnological reasons, because glycine betaine is one among a limited number of compatible solutes used by cells to counteract osmotic pressure. From a fundamental standpoint, choline oxidase has emerged as one of the paradigm enzymes for the oxidation of alcohols catalyzed by flavoproteins. Mechanistic, structural, and computational studies have elucidated the mechanism of action of the enzyme from Arthrobacter globiformis at the molecular level. Both choline and oxygen access to the active site cavity are gated and tightly controlled. Amino acid residues involved in substrate binding, and their contribution, have been identified. The mechanism of choline oxidation, with a hydride transfer reaction, an asynchronous transition state, the formation and stabilization of an alkoxide transient species, and a quantum mechanical mode of reaction, has been elucidated. The importance of nonpolar side chains for oxygen localization and of the positive charge harbored on the substrate for activation of oxygen for reaction with the reduced flavin have been recognized. Interesting phenomena, like the formation of a metastable photoinduced flavin-protein adduct, the reversible formation of a bicovalent flavoprotein, and the trapping of the enzyme in inactive conformations, have been described. This review summarizes the current status of our understanding on the structure-function-dynamics of choline oxidase.
Substitutions of S101 decrease proton and hydride transfers in the oxidation of betaine aldehyde by choline oxidase
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Choline oxidase oxidizes choline to glycine betaine, with two flavin-mediated reactions to convert the alcohol substrate to the carbon acid product. Proton abstraction from choline or hydrated betaine aldehyde in the wild-type enzyme occurs in the mixing time of the stopped-flow spectrophotometer, thereby precluding a mechanistic investigation. Mutagenesis of S101 rendered the proton transfer reaction amenable to study. Here, we have investigated the aldehyde oxidation reaction catalyzed by the mutant enzymes using steady-state and rapid kinetics with betaine aldehyde. Stopped-flow traces for the reductive half-reaction of the S101T/V/C variants were biphasic, corresponding to the reactions of proton abstraction and hydride transfer. In contrast, the S101A enzyme yielded monophasic traces like wild-type choline oxidase. The rate constants for proton transfer in the S101T/C/V variants decreased logarithmically with increasing hydrophobicity of residue 101, indicating a behavior different from that seen previously with choline for which no correlation was determined. The rate constants for hydride transfer also showed a logarithmic decrease with increasing hydrophobicity at position 101, which was similar to previous results with choline as a substrate for the enzyme. Thus, the hydrophilic character of S101 is necessary not only for efficient hydride transfer but also for the proton abstraction reaction.
Evidence for proton tunneling and a transient covalent flavin-substrate adduct in choline oxidase S101A
2017, Biochimica et Biophysica Acta - Proteins and Proteomics
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The kobs values for anaerobic flavin reduction were determined in a stopped-flow spectrophotometer by mixing the enzyme with choline at pL 10.0 and 15 °C and monitoring absorbance changes over time at 450 nm. pH 10 has previously been shown to be in the pH independent region for the WT CHO [48,50,54] and a number of enzyme variants with mutations in the active site, including E312D [45], H351A [44], H466A [56], H99N [21], and the S101A enzyme (data not shown). Anaerobic mixing of the enzyme with choline in buffer prepared with H2O resulted in the two-electron reduction of the enzyme-bound flavin, following a biphasic process (Fig. 2A).
The effect of temperature on the reaction of alcohol oxidation catalyzed by choline oxidase was investigated with the S101A variant of choline oxidase. Anaerobic enzyme reduction in a stopped-flow spectrophotometer was biphasic using either choline or 1,2-[²H₄]-choline as a substrate. The limiting rate constants k_lim1 and k_lim2 at saturating substrate were well separated (k_lim1/k_lim2 > 9), and were > 15-fold slower than for wild-type choline oxidase. Solvent deuterium kinetic isotope effects (KIEs) ~ 4 established that k_lim1 probes the proton transfer from the substrate hydroxyl to a catalytic base. Primary substrate deuterium KIEs ≥ 7 demonstrated that k_lim2 reports on hydride transfer from the choline alkoxide to the flavin. Between 15 °C and 39 °C the k_lim1 and k_lim2 values increased with increasing temperature, allowing for the analyses of H⁺ and H^– transfers using Eyring and Arrhenius formalisms. Temperature-independent KIE on the k_lim1 value (^H2Ok_lim1/^D2Ok_lim1) suggests that proton transfer occurs within a highly reorganized tunneling-ready-state with a narrow distribution of donor-acceptor distances. Eyring analysis of the k_lim2 value gave lines with the slope_(choline) > slope_(D-choline), suggesting kinetic complexity. Spectral evidence for the transient occurrence of a covalent flavin-substrate adduct during the first phase of the anaerobic reaction of S101A CHO with choline is presented, supporting the notion that an important role of amino acid residues in the active site of flavin-dependent enzymes is to eliminate alternative reactions of the versatile enzyme-bound flavin for the reaction that needs to be catalyzed.
Radiosynthesis and pre-clinical evaluation of [18F]fluoro-[1,2-2H4]choline
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Choline radiotracers are widely used for clinical PET diagnosis in oncology. [¹¹C]Choline finds particular utility in the imaging of brain and prostate tumor metabolic status, where 2-[¹⁸F]fluoro-2-deoxy-d-glucose (‘FDG’) shows high background uptake. More recently we have extended the clinical utility of [¹¹C]choline to breast cancer where radiotracer uptake correlates with tumor aggressiveness (grade). In the present study, a new choline analog, [¹⁸F]fluoro-[1,2-²H₄]choline, was synthesized and evaluated as a potential PET imaging probe.
[¹⁸F]Fluorocholine, [¹⁸F]fluoro-[1-²H₂]choline and [¹⁸F]fluoro-[1,2-²H₄]choline were synthesized by alkylation of the relevant precursor with [¹⁸F]fluorobromomethane or [¹⁸F]fluoromethyl tosylate. Radiosynthesis of [¹⁸F]fluoromethyl tosylate required extensive modification of the existing method. [¹⁸F]Fluorocholine and [¹⁸F]fluoro-[1,2-²H₄]choline were then subjected to in vitro oxidative stability analysis in a chemical oxidation model using potassium permanganate and an enzymatic model using choline oxidase. The two radiotracers, together with the corresponding di-deuterated compound, [¹⁸F]fluoro-[1-²H₂]choline, were then evaluated in vivo in a time-course biodistribution study in HCT-116 tumor-bearing mice.
Alkylation with [¹⁸F]fluoromethyl tosylate proved to be the most reliable radiosynthetic route. Stability models indicate that [¹⁸F]fluoro-[1,2-²H₄]choline possesses increased chemical and enzymatic (choline oxidase) oxidative stability relative to [¹⁸F]fluorocholine. The distribution of the three radiotracers, [¹⁸F]fluorocholine, [¹⁸F]fluoro-[1-²H₂]choline and [¹⁸F]fluoro-[1,2-²H₄]choline, showed a similar uptake profile in most organs. Crucially, tumor uptake of [¹⁸F]fluoro-[1,2-²H₄]choline was significantly increased at late time points compared to [¹⁸F]fluorocholine and [¹⁸F]fluoro-[1-²H₂]choline.
Stability analysis and biodistribution suggest that [¹⁸F]fluoro-[1,2-²H₄]choline warrants further in vivo investigation as a PET probe of choline metabolism.
Aryl-alcohol oxidase involved in lignin degradation. A mechanistic study based on steady and pre-steady state kinetics and primary and solvent isotope effects with two alcohol substrates
2009, Journal of Biological Chemistry
Citation Excerpt :
However, in AAO oxidation of p-anisyl alcohol the D(Vmax/Km) value obtained was lower than those of DVmax and Dkred, and the cleavage of the CH bond does not appear to be the only rate-limiting step for catalysis. Stronger masking of bond cleavage has been found for cholesterol oxidase and glucose oxidase, with the reported KIE (around 2–3) significantly lower than their intrinsic values (32, 35, 36), whereas large substrate KIE values have been found for choline oxidase (27, 37). The complexities described made difficult an unambiguous determination of the catalytic mechanisms of the two former oxidases, while that of choline oxidase has been investigated during recent years as a model flavooxidase (38).
Aryl-alcohol oxidase (AAO) is a FAD-containing enzyme in the GMC (glucose-methanol-choline oxidase) family of oxidoreductases. AAO participates in fungal degradation of lignin, a process of high ecological and biotechnological relevance, by providing the hydrogen peroxide required by ligninolytic peroxidases. In the Pleurotus species, this peroxide is generated in the redox cycling of p-anisaldehyde, an extracellular fungal metabolite. In addition to p-anisyl alcohol, the enzyme also oxidizes other polyunsaturated primary alcohols. Its reaction mechanism was investigated here using p-anisyl alcohol and 2,4-hexadien-1-ol as two AAO model substrates. Steady state kinetic parameters and enzyme-monitored turnover were consistent with a sequential mechanism in which O₂ reacts with reduced AAO before release of the aldehyde product. Pre-steady state analysis revealed that the AAO reductive half-reaction is essentially irreversible and rate limiting during catalysis. Substrate and solvent kinetic isotope effects under steady and pre-steady state conditions (the latter showing ∼9-fold slower enzyme reduction when α-bideuterated substrates were used, and ∼13-fold slower reduction when both substrate and solvent effects were simultaneously evaluated) revealed a synchronous mechanism in which hydride transfer from substrate α-carbon to FAD and proton abstraction from hydroxyl occur simultaneously. This significantly differs from the general mechanism proposed for other members of the GMC oxidoreductase family that implies hydride transfer from a previously stabilized substrate alkoxide.
Effect of a conservative mutation of an active site residue involved in substrate binding on the hydride tunneling reaction catalyzed by choline oxidase
2009, Archives of Biochemistry and Biophysics
Citation Excerpt :
For comparison, a previous investigation of the temperature dependence of the reductive half-reaction with the wild-type enzyme using a steady state approach yielded slopes that were similar to one another with choline and 1,2-[2H4]-choline, corresponding to ΔH‡ values of ∼18 kJ mol−1[7]. The comparison of the pre-steady state data for the Glu312Asp enzyme with the steady state data for the wild-type is based on previous results that are consistent with the hydride transfer step being rate-limiting in the reductive half-reaction catalyzed by wild-type choline oxidase [10,11]. Similar entropies of activation (−TΔS‡) of ∼40 kJ mol−1 were observed with choline and 1,2-[2H4]-choline as substrate for the Glu312Asp enzyme, yielding an isotope effect on the Eyring intercept of ∼0.9.
The reaction of alcohol oxidation catalyzed by choline oxidase was previously shown to occur through the tunneling of a hydride ion from the α-carbon of an activated alkoxide species to the N(5) atom of FAD within a highly preorganized enzyme-substrate complex [G. Gadda, Biochemistry 47 (2008) 13745–13753]. In the present study, a glutamate residue (312) that participates in substrate binding has been replaced with aspartate by site-directed mutagenesis, and the effect of the substitution on the kinetic parameters of the enzyme and the mechanism of hydride ion transfer were investigated using stopped-flow spectrophotometry. The thermodynamic parameters associated with the enzymatic reaction catalyzed by the mutant enzyme and the temperature dependence of the kinetic isotope effects are consistent with a hydride transfer reaction occurring either through environmentally assisted tunneling or a classical over-the-barrier transition state, but not within a preorganized enzyme-substrate complex. In contrast, less than threefold changes in the K_d values for choline were observed in the mutant enzyme with respect to the wild-type enzyme. Thus, the conservative substitution of an active site residue that is involved in substrate binding, but not directly in catalysis, affects primarily the k_red value that reports on catalysis and minimally the K_d value that reports on binding, thereby providing an example of the interplay that these kinetic parameters may have in enzymatic reactions.

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pH and deuterium kinetic isotope effects studies on the oxidation of choline to betaine-aldehyde catalyzed by choline oxidase

Abstract

Introduction

Section snippets

Materials

pH dependence of the V/K values for choline and [1,2-2H4]-choline as substrate

Discussion

Acknowledgements

J. Mol. Biol.

Arch. Biochem. Biophys.

Biochim. Biophys. Acta

J. Biol. Chem.

Arch. Biochem. Biophys.

Biosens. Bioelectron.

FEMS Microbiol. Lett.

Bioorg. Med. Chem. Lett.

Biochim. Biophys. Acta

J. Biol. Chem.

Bioorg. Med. Chem. Lett.

Bioorg. Chem.

Biosens. Bioelectron.

Tetrahedron Lett.

J. Biol. Chem.

J. Biochem. (Tokyo)

Biochem. J.

Eur. J. Biochem.

Eur. J. Biochem.

Biochem. J.

Eur. J. Biochem.

Biochemistry

Biochemistry

Adv. Exp. Med. Biol.

J. Biochem. (Tokyo)

Biochemistry

Biochemistry

pH dependence of the V/K values for choline and [1,2-²H₄]-choline as substrate