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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• 作者：Mylrajan Muthusamy ; Matthew R. Burrell ; Roger N. F. Thorneley ; Stephen Bornemann
• 刊名：Biochemistry
• 出版年：2006
• 出版时间：September 5, 2006
• 年：2006
• 卷：45
• 期：35
• 页码：10667 - 10673
• 全文大小：113K
• 年卷期：v.45,no.35(September 5, 2006)
• ISSN：1520-4995

文摘

Oxalate decarboxylase converts oxalate to formate and carbon dioxide and uses dioxygen asa cofactor despite the reaction involving no net redox change. We have successfully used Fourier transforminfrared spectroscopy to monitor in real time both substrate consumption and product formation for thefirst time. The assignment of the peaks was confirmed using [¹³C]oxalate as the substrate. The K_m foroxalate determined using this assay was 3.8-fold lower than that estimated from a stopped assay. Theinfrared assay was also capable of distinguishing between oxalate decarboxylase and oxalate oxidaseactivity by the lack of formate being produced by the latter. In D₂O, the product with oxalate decarboxylasewas 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 V_max (7.1 ± 0.3), K_m for oxalate (3.9 ±0.9), and k_cat/K_m (1.8 ± 0.4) indicative of a proton transfer event during a rate-limiting step. Semiempiricalquantum mechanical calculations on the stability of formate-derived species gave an indication of thestability and nature of a likely enzyme-bound formyl radical catalytic intermediate. The capability of theenzyme to bind formate under conditions in which the enzyme is known to be active was determined byelectron paramagnetic resonance. However, no enzyme-catalyzed exchange of the C-hydron of formatewas observed using the infrared assay, suggesting that a formyl radical intermediate is not accessible inthe reverse reaction. This restricts the formation of potentially harmful radical intermediates to the forwardreaction.