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

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posted on 26.01.2016, 00:00 by Ebenezer Joseph, Cuong Quang Le, Toan Nguyen, Mercy Oyugi, Mohammad Shawkat Hossain, Frank W. Foss, Kayunta Johnson-Winters
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.

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