Evidence of Negative Cooperativity and Half-Site Reactivity within an F420-Dependent Enzyme: Kinetic Analysis of F420H2:NADP+ Oxidoreductase

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• 作者：Ebenezer Joseph ; Cuong Quang Le ; Toan Nguyen ; Mercy Oyugi ; Mohammad Shawkat Hossain ; Frank W. Foss Jr. ; Kayunta Johnson-Winters
• 刊名：Biochemistry
• 出版年：2016
• 出版时间：February 23, 2016
• 年：2016
• 卷：55
• 期：7
• 页码：1082-1090
• 全文大小：497K
• ISSN：1520-4995

文摘

Here, we report the very first example of half-site reactivity and negative cooperativity involving an important F₄₂₀ cofactor-dependent enzyme. F₄₂₀H₂:NADP⁺ oxidoreductase (Fno) is an F₄₂₀ cofactor-dependent enzyme that catalyzes the reversible reduction of NADP⁺ through the transfer of a hydride from the reduced F₄₂₀ cofactor. These catalytic processes are of major significance in numerous biochemical processes. While the steady-state kinetic analysis showed classic Michaelis–Menten kinetics with varying concentrations of the F₄₂₀ redox moiety, FO, such plots revealed non-Michaelis–Menten kinetic behavior when NADPH was varied. The double reciprocal plot of the varying concentrations of NADPH displays a downward concave shape, suggesting that negative cooperativity occurs between the two identical monomers. The transient state kinetic data show a burst prior to entering steady-state turnover. The burst suggests that product release is rate-limiting, and the amplitude of the burst phase corresponds to production of product in only one of the active sites of the functional dimer. These results suggest either half-site reactivity or an alternate sites model wherein the reduction of the cofactor, FO occurs at one active site at a time followed by reduction at the second active site. Thus, the data imply that Fno may be a functional regulatory enzyme.