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Real-Time Monitoring of the Oxalate Decarboxylase Reaction and Probing Hydron Exchange in the Product, Formate, Using Fourier Transform Infrared Spectroscopy

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    Real-Time Monitoring of the Oxalate Decarboxylase Reaction and Probing Hydron Exchange in the Product, Formate, Using Fourier Transform Infrared Spectroscopy†
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    Biological Chemistry Department, John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, United Kingdom
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    Biochemistry

    Cite this: Biochemistry 2006, 45, 35, 10667–10673
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    https://doi.org/10.1021/bi060460q
    Published August 8, 2006
    Copyright © 2006 American Chemical Society
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    Abstract

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    Oxalate decarboxylase converts oxalate to formate and carbon dioxide and uses dioxygen as a cofactor despite the reaction involving no net redox change. We have successfully used Fourier transform infrared spectroscopy to monitor in real time both substrate consumption and product formation for the first time. The assignment of the peaks was confirmed using [13C]oxalate as the substrate. The Km for oxalate determined using this assay was 3.8-fold lower than that estimated from a stopped assay. The infrared assay was also capable of distinguishing between oxalate decarboxylase and oxalate oxidase activity by the lack of formate being produced by the latter. In D2O, the product with oxalate decarboxylase was C-deuterio formate rather than formate, showing that the source of the hydron was solvent as expected. Large solvent deuterium kinetic isotope effects were observed on Vmax (7.1 ± 0.3), Km for oxalate (3.9 ± 0.9), and kcat/Km (1.8 ± 0.4) indicative of a proton transfer event during a rate-limiting step. Semiempirical quantum mechanical calculations on the stability of formate-derived species gave an indication of the stability and nature of a likely enzyme-bound formyl radical catalytic intermediate. The capability of the enzyme to bind formate under conditions in which the enzyme is known to be active was determined by electron paramagnetic resonance. However, no enzyme-catalyzed exchange of the C-hydron of formate was observed using the infrared assay, suggesting that a formyl radical intermediate is not accessible in the reverse reaction. This restricts the formation of potentially harmful radical intermediates to the forward reaction.

    Copyright © 2006 American Chemical Society

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    †

     This research was supported by the Biotechnology and Biological Sciences Research Council with a Core Strategic Grant for the John Innes Centre and a Biochemistry & Cell Biology Committee Studentship for M.R.B.

    ‡

     Contributed equally to this work.

    *

     Correspondence should be addressed to this author. Tel:  +44 1603 450741. Fax:  +44 1603 450018. E-mail:  stephen.bornemann@ bbsrc.ac.uk.

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    This article is cited by 18 publications.

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    Biochemistry

    Cite this: Biochemistry 2006, 45, 35, 10667–10673
    Click to copy citationCitation copied!
    https://doi.org/10.1021/bi060460q
    Published August 8, 2006
    Copyright © 2006 American Chemical Society
    Request reuse permissions

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