Human Metabolism. Keith N. Frayn

of the TCA cycle does not have sufficient energy to reduce NAD+ or FAD but does have sufficient energy to phosphorylate GDP to GTP. One acetyl-CoA molecule reduces one FAD and three NAD⁺ molecules.

      The stoichiometry is precise, such that both the carbons of the acetyl group are oxidised (though as shown by radiolabelling experiments, not the actual two carbon atoms that entered the cycle), hence all their useful energy is extracted, resulting in two carbon dioxide molecules as ‘waste’ products. The NADH and FADH₂ will subsequently be re-oxidised by passing on the electron(s) they carry down the redox proteins of the electron transport chain, regenerating NAD⁺ and FAD in the process for further electron carriage; by coupling this sequential oxidation-reduction to phosphorylation of ADP to ATP (‘oxidative phosphorylation’), a common energy carrier for diverse cellular functions (ATP) is synthesised (see below).

An important point about the TCA cycle is that its intermediates are not specifically dedicated to the cycle – they are also used for other purposes in several other metabolic pathways (e.g. amino acid metabolism; porphyrin synthesis; purine nucleotide metabolism). Processes which utilise TCA cycle intermediates and hence deplete them are termed ‘cataplerotic’ whilst processes which replenish them are termed ‘anaplerotic.’ In order for a substance to be anaplerotic it must have more than two carbons (or equivalent): acetyl groups cannot replenish the cycle because both their carbons (equivalent) are oxidised by the cycle (2 × CO₂) and no net gain of carbon occurs. Since fatty acids represent a chain of 2-carbon groups (they are, effectively, a storage form of acetyl groups – hence their mostly even numbers of carbons), when fatty acids are used for energy (e.g. during starvation) they are broken back down to 2-carbon acetyl groups (by β-oxidation) and cannot therefore be anaplerotic – indeed reliance on fatty acids for oxidation does lead to cataplerosis: if a cell were to derive its energy entirely from fatty acids, eventually cataplerotic depletion of TCA cycle intermediates would occur due to ongoing utilisation of the intermediates by other pathways, combined with the inability of the fatty acids (acetyl groups) to ‘top up’ the same cycle intermediates. The same applies for ketone bodies (acetoacetate, 3-hydroxybutyrate) which are 4-carbon compounds but effectively represent two acetyl groups joined together – a transport form of acetyl-CoA; their utilisation first requires them to be converted back into acetyl-CoA, and they cannot enter the TCA cycle as 4-carbon compounds. Anaplerosis has to occur from carbohydrates or (glucogenic) amino acids, which can bypass the PDH step and insert 4-carbon intermediates (e.g. oxaloacetate; derived from pyruvate) into the cycle. The importance of these anaplerotic pathways, and the enzymes that achieve them, to maintain cellular metabolic efficiency is now well recognised. See later, Box 5.3, for more information.

      1.3.1.5 Electron transport chain

      The final stage of energy production is the synthesis of ATP by phosphorylation of ADP, coupled to the re-oxidation of NADH and FADH₂ to permit further electron carriage to occur (oxidative phosphorylation). The energy is now carried in the phosphoanhydride bonds of the phosphate groups of ATP; these are commonly called ‘high energy’ bonds (see Box 1.6).

Box 1.6 High-energy bonds

      The term ‘high-energy bond’ is not strictly accurate. They are not ‘special’ bonds, but rather they release their free energy when hydrolysed. They may be denoted ∼, and it can be seen that ADP can act as a source of energy when its terminal phosphate group is hydrolysed to AMP.

       AMP: Ad-P

       ADP: Ad-P∼P

       ATP: Ad-P∼P∼P

      ADP has a special role in bioenergetics because beside acting as a (limited) energy source in its own right, its availability is one factor that regulates the rate of ATP synthesis.

      A close structural analogue of adenosine triphosphate (ATP) is guanosine triphosphate (GTP) which also carries energy. The presence of these two forms of energy carriage likely represents a form of metabolic compartmentation, separating pathways by their molecular preferences. GTP also has a function in regulation, especially in signal transduction involving G-proteins.

The energy for the phosphorylation is derived from the electrons contained in the reduced forms of the electron carriers. As the electron is passed sequentially down a series of carriers, the released energy is harnessed. The mechanism responsible is the electron transport chain, located in the inner mitochondrial membrane (IMM). The IMM is a highly specialised membrane which is extremely selectively impermeable. It comprises four protein complexes (complex I, II, III, IV) together with a loosely associated cytochrome (cytochrome c) and the (non-protein) quinone CoQ10 (ubiquinone). All these components have variable redox states and act as electron acceptors (oxidising agents) and electron donors (reducing agents); they can function as electron carriers by shifting between their reduced (electron containing) and oxidised (electron deficient) forms. To facilitate this, the proteins contain transition metals (Fe, Cu), complexed in prosthetic groups (e.g. haem) or complexed to sulphur (Fe-S centres) which are readily capable of gaining or losing an electron. They are organised in a sequential arrangement within the IMM such that the electrons are passed down a gradual incremental energy gradient. Complex I oxidises NADH back to NAD⁺, whilst complex II oxidises FADH₂ back to FAD, both passing electrons (as pairs) to CoQ10, thence to complex III, then cytochrome c, and eventually to complex IV. Complex IV, the final redox protein in the chain, combines the electrons it receives with molecular oxygen (O₂, the final electron acceptor), reducing it to water. Complexes I, III, and IV use the energy of electron transfer to pump protons (H⁺) out of the mitochondrial matrix across the IMM and into the intermembrane space beyond. This creates an electrochemical gradient between the inside and outside of the mitochondrion. The energy of this H⁺ gradient is finally utilised to drive phosphorylation of ADP to ATP: the protons can only re-enter the mitochondrial matrix by passing through ATP synthase (Fo/F₁ ATPase; also called complex V although it is not a part of the actual electron transport chain), another IMM-spanning protein. Proton passage through this large protein complex provides the energy for ATP synthesis (the chemiosmotic process). Hence, provided the IMM is otherwise impermeable to protons, the oxidation of NADH/FADH₂ by electron transport is tightly coupled to the phosphorylation of ADP to ATP. However, if protons leak through the IMM back into the mitochondrial matrix, the gradient is dissipated (‘uncoupled’) and ATP synthesis cannot occur, leading to mitochondrial inefficiency. The final step in metabolism is the export of ATP out of the mitochondrion and into the cytosol, and this is achieved by an adenine nucleotide translocator also spanning the IMM.

      In the remainder of this chapter, we will outline the major metabolic pathways of ‘energy metabolism’ that will be considered further in this book, signposting the reader to where these are discussed in more detail.

      1.3.2 Carbohydrate metabolism

      1.3.2.1 Pathways of glucose metabolism

Carbohydrate metabolism centres around the hexose sugar glucose (Figure 1.7). Glucose is a ubiquitous sugar which may be derived from dietary carbohydrate or synthesised in the body. It is stored in polymeric form as glycogen: this removes the osmotic problems that would arise if it were stored in cells as free sugars (Section 1.2.2.1). Glucose, as a polar molecule, cannot cross membranes composed of phospholipid bilayers by diffusion, and two families of glucose transporter proteins (GLUTs, and sodium-glucose linked transporters, SGLTs: see Chapter 2, Box 2.2), expressed in a tissue-specific manner, facilitate its rapid movement in and out of cells. The pathways of glucose metabolism inside the cell are illustrated in simplified form in Figure 1.14, which shows the key steps of the various pathways responsible for its utilisation, disposal and production. Glucose can be stored as glycogen, used to provide 5-carbon sugars and NADPH for,