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

Abstract

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.

Effects of isoleucine 135 side chain length on the cofactor donor-acceptor distance within F420H2:NADP+ oxidoreductase: A kinetic analysis

“…The FO and NADPH binding experiments have been reported previously for wt Fno [16] . The binding experiments for the Fno variants were conducted in a similar manner as wt Fno for comparison and are described here.…”

“…The binding assay was monitored as described above for the FO binding studies. A decrease in tryptophan emission at 340 nm was observed for both FO and NADPH titrations and used for calculation of the dissociation constants [16] , [17] . A plot of change in fluorescence vs .…”

“…The rapid kinetic experiments of Fno variants were conducted in a similar manner as our previous studies of wt Fno [16] . These experiments were performed using a Hitech Scientific DX2 stopped-flow spectrophotometer at 22 °C.…”

“…When varying Fno concentrations, the amplitude of the burst phase corresponded to only ~50% FO reduction. This behavior suggests half-site reactivity [16] . Half-site reactivity has not been seen previously with Fno, or other F 420 cofactor dependent enzymes, thus providing potential valuable new insights into enzymes that use this unique cofactor.…”

Chloride promotes refolding of active Vibrio alkaline phosphatase through an inactive dimeric intermediate with an altered interface

“…The benefit of homodimer formation has been attributed to several factors, including increased stability by formation of interface contacts [1,2], extra sites becoming available for ligand binding [3], faster folding being facilitated that minimizes occurrence of misfolded intermediates [4] and the provision of mechanisms for allosteric regulation [5]. Many homodimeric enzymes show evidence of cooperativity [6][7][8][9][10][11] and especially negative cooperativity resulting in half-ofsites reactivity [8,10]. Thus, the role of having two identical subunits is in many cases to increase the conformational space through a dynamical asymmetry, coordinated by interface contacts [12][13][14].…”