biostudies-literatureLe CQNational Institutes of HealthNIGMS NIH HHS114-120https://www.ebi.ac.uk/biostudies/studies/S-EPMC5614548biostudies-literatureUnknown9F₄₂₀H₂:NADP⁺ Oxidoreductase (Fno) catalyzes the reversible reduction of NADP⁺ to NADPH by transferring a hydride from the reduced F₄₂₀ cofactor. Here, we have employed binding studies, steady-state and pre steady-state kinetic methods upon <i>wt</i>Fno and isoleucine 135 (I135) Fno variants in order to study the effects of side chain length on the donor-acceptor distance between NADP⁺ and the F₄₂₀ precursor, FO. The conserved I135 residue of Fno was converted to a valine, alanine and glycine, thereby shortening the side chain length. The steady-state kinetic analysis of <i>wt</i>Fno and the variants showed classic Michaelis-Menten kinetics with varying FO concentrations. The data revealed a decreased <i>k</i>_cat as side chain length decreased, with varying FO concentrations. The steady-state plots revealed non-Michaelis-Menten kinetic behavior when NADPH was varied. The double reciprocal plot of the varying NADPH concentrations displays a downward concave shape, while the NADPH binding curves gave Hill coefficients of less than 1. These data suggest that negative cooperativity occurs between the two identical monomers. The pre steady-state Abs₄₂₀ versus time trace revealed biphasic kinetics, with a fast phase (hydride transfer) and a slow phase. The fast phase displayed an increased rate constant as side chain length decreased. The rate constant for the second phase, remained ~2 s^-1 for each variant. Our data suggest that I135 plays a key role in sustaining the donor-acceptor distance between the two cofactors, thereby regulating the rate at which the hydride is transferred from FOH₂ to NADP⁺. Therefore, Fno is a dynamic enzyme that regulates NADPH production.Biochemistry and biophysics reportsEffects of isoleucine 135 side chain length on the cofactor donor-acceptor distance within F₄₂₀H₂:NADP⁺ oxidoreductase: A kinetic analysis.PMC56145481 R15 GZM113223 - 01AR15 GM113223Phan TAubert JNguyen TLe CQJohnson-Winters KUllah MHOyugi MTran JJoseph EfalseEffects of isoleucine 135 side chain length on the cofactor donor-acceptor distance within F₄₂₀H₂:NADP⁺ oxidoreductase: A kinetic analysis.F₄₂₀H₂:NADP⁺ Oxidoreductase (Fno) catalyzes the reversible reduction of NADP⁺ to NADPH by transferring a hydride from the reduced F₄₂₀ cofactor. Here, we have employed binding studies, steady-state and pre steady-state kinetic methods upon <i>wt</i>Fno and isoleucine 135 (I135) Fno variants in order to study the effects of side chain length on the donor-acceptor distance between NADP⁺ and the F₄₂₀ precursor, FO. The conserved I135 residue of Fno was converted to a valine, alanine and glycine, thereby shortening the side chain length. The steady-state kinetic analysis of <i>wt</i>Fno and the variants showed classic Michaelis-Menten kinetics with varying FO concentrations. The data revealed a decreased <i>k</i>_cat as side chain length decreased, with varying FO concentrations. The steady-state plots revealed non-Michaelis-Menten kinetic behavior when NADPH was varied. The double reciprocal plot of the varying NADPH concentrations displays a downward concave shape, while the NADPH binding curves gave Hill coefficients of less than 1. These data suggest that negative cooperativity occurs between the two identical monomers. The pre steady-state Abs₄₂₀ versus time trace revealed biphasic kinetics, with a fast phase (hydride transfer) and a slow phase. The fast phase displayed an increased rate constant as side chain length decreased. The rate constant for the second phase, remained ~2 s^-1 for each variant. Our data suggest that I135 plays a key role in sustaining the donor-acceptor distance between the two cofactors, thereby regulating the rate at which the hydride is transferred from FOH₂ to NADP⁺. Therefore, Fno is a dynamic enzyme that regulates NADPH production.2017-01-01T00:00:00Z2017 Mar2024-11-08T21:22:49.592Z2019-03-27T02:57:23ZS-EPMC56145482895599510.1016/j.bbrep.2016.11.012