How is NADPH produced in (a) Photosynthetic AND (b) Non-photosynthetic cells? How and where are ketone bodies (a) synthesised AND (b) utilised?
How is NADPH produced in (a) Photosynthetic AND (b) Non-photosynthetic cells? Give examples of how NADPH may be used in each case.
NADPH is produced in photosynthetic cells by the light-dependent reactions of photosynthesis in order to be used for reduction of carbon atoms in the light-independent stage, or the Calvin Cycle.
NADPH is produced in the light reactions which convert light energy into chemical energy. Photons absorbed by photosystem II in the thylakoid membrane, and the energy is used to break down water and release two electrons which are excited and begin to move along an electron transport chain in the membrane. As they move along, they release energy and pass through cytochrome bf, which is used to generate a proton-motive force and generate ATP until the electrons reach photosystem I. Photosystem I absorbs photons with wavelength 700nm and uses the energy to excite the electrons again, so that they can move further along the transport chain and eventually combine with NADP+ and H+ to give NADPH.
NADPH is used in the Calvin cycle, which takes place in the stroma of chloroplasts and synthesises hexoses from carbon dioxide and water. In order to oxidise NADPH by this process, ribulose 5-phosphate is first phosphorylated by R5P kinase to generate ribulose-1,5-bisphosphate. This is carboxylated and broken down by CO2 to give two molecules of 3-phosphoglycerate in a reaction catalysed by the enzyme Rubisco, and which features two intermediates – one highly unstable and the other an enediolate. The enediolate forms first as the ketone group on R-1,5-BP is reduced, before carboxylate adds to form the unstable intermediate which immediately breaks down via hydrolysis to give two 3-phosphoglycerates. Each of these is then phosphorylated by ATP with 3-PG kinase to give 1,3-bisphosphoglycerate. It is at this stage that NADPH is involved in the reaction, as it donates a proton to a 1,3-BPG so that it will reduce and lose Pi to give glyceraldehyde 3-phosphate. Two G3P molecules can then combine to regenerate the R5P at the start of the cycle so that it can restart.
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However, in non-photosynthetic cells the process is different. One way to generate NADPH is the pentose phosphate pathway. The pentose phosphate pathway begins with the oxidation of glucose 6-phosphate (G6P) by G6P dehydrogenase, which protonates NADP+ to give 6-phosphoglucono-1,5-lactone. It also results in generation of NADPH almost immediately after the start of the pathway. The gluconolactonase converts this to 6-phosphogluconate by releasing a proton from water, before 6-phosphogluconate dehydrogenase converts this to ribulose-5-phosphate. This stage also forms NADPH, as well as carbon dioxide. The reaction pathway continues towards the 3-carbon sugars of glycolysis, but with no more generation of NADPH. This process is not only needed in non-photosynthetic cells, but also occurs in photosynthetic cells and even often in the chloroplasts of these cells where photosynthesis also occurs, or other plastids within plant cells.
Furthermore, the transmembrane movement of malate and certain amino acids can also generate NADPH.
How and where are ketone bodies (a) synthesised AND (b) utilised?
Ketone bodies are primarily produced in the mitochondria of liver cells. They are synthesised in order to be transported for transport of acetyl-CoA so that all of these acetyl units can be oxidised – for example, during starvation, gluconeogenesis depletes the supply of oxaloacetate which means that it cannot be used to combine with acetyl-CoA. Therefore, acetyl-CoA resulting from fatty acid breakdown must be oxidised in another way, in this case by conversion to ketone bodies. The ketone bodies are acetone, D-3-hydroxybutyrate and acetoacetate, and these are three water-soluble molecules which can pass through the blood-brain barrier (which acetyl-CoA cannot) and be used to make long-chain fatty acids in the brain cells. Ketone bodies are formed when fatty acids are released by adipose tissue to be transported to the liver, where they are first converted to acetyl-CoA in the mitochondria and then exported as acetoacetate.
Three steps convert acetyl-CoA to acetoacetate. First, acetoacetyl-CoA is formed via condensation of two acetyl-CoA molecules, releasing CoA in a reaction which is catalysed by 3-thiolase – an enzyme which catalyses bond splitting and forming by sulphur. This reaction has an unfavourable equilibrium and is driven by the highly favourable equilibrium of the next reaction. Another acetyl-CoA, again, reacts with this product as well as water, releasing the CoA from the acetyl-CoA to dorm 3-hydroxy-3-methyl-glutaryl CoA (HMG-CoA) by action of HMG-CoA synthase. This product is then cleaved to give acetoacetate, one of the three ketone bodies, and acetyl-CoA. Therefore, the sum of these reactions is as follows:
2Acetyl-CoA + H2O → Acetoacetate + 2CoA + H+
Acetoacetate can be and is often used in order to form the other two ketone bodies, acetone and D-3-hydroxybutyrate (D3HB). It can be reduced in the mitochondrial matrix by D3HB dehydrogenase, which oxidises NADH in the process and so therefore the rate of formation and the ratio of acetoacetate/D3HB concentrations in the mitochondria depends largely on the NADH/NAD+ ratio.
Furthermore, acetoacetate can also decarboxylate to give acetone, the third ketone body, but this spontaneous process is slow and only occurs gradually over time.
Utilisation of ketone bodies occurs as they are the major fuel in some tissues. After synthesis in the liver mitochondria, they diffuse into the blood to be transported to other tissues such as the heart - in which acetoacetate is actually preferred to glucose as a fuel. This is generally not true in the brain, however, and so ketone bodies are only required in the brain in great concentrations when the body is glucose-deficient i.e. during times of starvation, but also as the primary source of energy in the renal cortex. It is in these cells that ketone bodies are converted to acetyl-CoA so that this can enter the TCA cycle. This conversion occurs when acetoacetate receives a CoA group from succinyl-CoA via catalysis by CoA transferase to give acetoacyl-CoA. This is then cleaved by thiolase to yield two molecules of acetyl-CoA, as this is the molecule needed for entry into the TCA cycle in order to generate NADH and FADH for use in oxidative phosphorylation to generate ATP. When the demand for acetyl-CoA becomes high in these cells, ketone bodies are transported to them instead of acetyl-CoA because the CoA transferase enzyme is not present in liver mitochondria so this must occur in the energy-requiring cell. Therefore, the ketone bodies act as a water-soluble, transportable form of acetyl units.