How Is NADH Converted into NADPH?
Reduced phosphorylated nicotinamide adenine dinucleotide (NADPH) is an essential coenzyme in anabolic biosynthetic reactions in cells. It also reduces glutathione, thus limiting damage from free radicals. NADPH is most commonly produced via the pentose phosphate pathway from glucose-6-phosphate and phosphorylated nicotinamide adenine dinucleotide (NADP+). It also can be produced directly from reduced nicotinamide adenine dinucleotide (NADH) in the presence of NADP+ using the enzyme nicotinomide nucleotide transhydrogenase (NNT) in a redox reaction. This reaction occurs in mitochondria and bacteria.
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Instructions
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Produce energy for the reaction. Because this is an endergonic reaction, it requires a source of energy. This energy comes from an electrochemical gradient created by a proton pump. The enzyme NNT is composed of two monomers (a dimer). Each monomer is composed of three subunits, called dI, dII, and dIII. The dI binds NADH, dIII binds NADP+ and dII spans the inner mitochondrial membrane. The dII has a central channel that pumps protons (hydrogen ions) from the cytoplasmic side to the matrix side, creating a gradient with a potential difference of 130-170 mV and a pH difference of 0.5 to 1 pH.
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Expose the binding sites of NNT, and bind the reactants. The energy you created previously is used to change the shape of NNT from an occluded conformation to an open conformation. In this conformation, the binding sites for NADH and NADP+ are at a distance from one another and can't exchange the hydride ion (H-), but they are available to the reactants NADH and NADP+ present in the solvent. The dI subunit binds NADH, and dIII binds NADP+.
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Transfer a hydride ion from NADH to NADP+. When dII stops pumping protons, the electrochemical gradient ceases. At neutral pH, the conformation of NNT changes back to the occluded state, moving the binding sites at dI and dIII closer together. This allows the transfer of the hydride ion from NADH to NADP+ to form new molecules of NADPH. The occluded conformation excludes interaction of the binding sites with the cytosol.
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Release the products, and bind new reactants. As the hydrogen ions are pumped back through the dII channel, a gradient once again forms, changing the conformation of NNT back to the open state. The products NADPH and NAD+ are released, and new reactants NADH and NADP+ bind. The activation of this pathway seems to be regulated by the concentration of NADPH in the solvent, though the exact mechanism is unknown.
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Tips & Warnings
The overall reaction is (NADH) + (NADP+) + (H+)out " (NAD+) + (NADPH) + (H+)in. The energy of the gradient drives the [NADPH][NAD+]/[NADP+][NADH] > 400, thus favoring the production of NADPH. Together with the two isocitrate dehydrogenases, a "mini cycle" between isocitrate and ∝ketoglutarate fine tunes the TCA (Krebs) cycle. The regulation of the proton pump is under study.
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