3 General Principles of Nutrient Metabolism

  • Understand the central role of ATP and NADH in metabolism
  • Understand the difference between phosphoryl transfer reactions and redox reactions
  • Understand the chemical activation of molecules
  • ATP hydrolysis, facilitation of reactions by ATP
  • Redox reaction and storage of reducing equivalents
  • Redox potential
  • Substrate-level phosphorylation
  • Chemiosmotic phosphorylation
  • Metabolic coupling
  • Use of thioesters
What the economy is to human society, metabolism is to the cell. In short, metabolism is the ensemble of chemical processes by which cells obtain the goods and services required for their continuance, growth and reproduction.
Franklin M. Harold
The Way of the Cell (p. 73). Oxford University Press. Kindle Edition.

This is a chapter that requires some chemistry background. If you need to brush up on nucleophilic and electrophilic centers and their reactivity, please watch this video.

In the previous chapter we learned that fast complete oxidation of nutrients (combustion) generates large amounts of energy. Combustion yields a lot of heat, but little is converted into useful work. We need to understand how our body breaks down nutrients without wasting all the energy as heat.

The key principle is to breakdown nutrients in small steps and to store the energy generated in particular steps. There are two main molecules that are used to store energy derived from biological reactions, namely nicotinamide dinucleotide (NAD) (and its cousin NADP) and adenosine triphosphate ATP (Fig. 1). 

Fig. 1: Molecules involved in energy transfer

There are more molecules involved in energy transfer, for instance GTP (phosphoryl transfer) and FAD (electron transfer), but the principles are the same as illustrated here. How do these molecules store energy? ATP is the major energy currency of the human body. It is used for muscle movement, the establishment of ion gradients and hence neural activity, the absorption of nutrients, and many metabolic processes wouldn’t occur without it. Its cousin GTP, which is generated by phosphorylation of GDP by ATP is central for protein biosynthesis. How can ATP store energy? In the previous chapter we learned that the free energy of a reaction depends on its distance from the equilibrium. This is the key to the ATP hydrolysis reaction. In our body, ATP and phosphate concentrations are typically around 5 mM, while ADP is less than 1 mM. If we would let the reaction come to its equilibrium we would have about 5 mM ADP and 5 mM phosphate, but only 0.0000005 mM ATP. It is the energy of life that keeps ATP at any time far out of equilibrium. After a stroke or heart attack, ATP concentrations sink rapidly, because of reduced blood flow to affected brain regions or heart tissue. This causes cell death. A biochemist could argue that a definition of life is to have more ATP than ADP, every known organism uses this system. 

As in economics, the linkage between supply and demand is intensely dynamic. The great highways of energetics, respiration and photosynthesis, keep the ATP/ADP ratio high, far away from equilibrium, and that, in turn, allows ATP to serve as an energy donor, displacing from equilibrium those reactions in which it participates. All biosynthetic processes, and also those that entail movement or transport, are energized either by ATP or by one of the more specialized energy carriers, and the latter are linked to the ATP/ADP couple, as it were by a system of exchange rates.
Harold, Franklin M.
The Way of the Cell (p. 42). Oxford University Press. Kindle Edition.

 There are two major ways to make ATP in our body. The first is a phosphoryl transfer reaction from a metabolic intermediate, the second is a direct condensation between phosphate and ADP to form ATP in a sophisticated nanomachine called the ATP synthase. Fig. 2 shows the Phosphoryl-transfer from a phosphorylated intermediate in the glycolysis pathway after initial oxidation.

Fig. 2 Preservation of oxidation energy through phosphoryl transfer onto ADP. The oxidation is highlighted by the circles. The phosphoryl transfer occurs in the subsequent reaction.

The oxidation allows condensation of phosphate to the newly generated carboxyl group. This phosphate group is then transferred to ADP forming ATP. Such a reaction is called substrate level phosphorylation. As a result, some of the oxidation energy is stored in the form of ATP. In fact, further rearrangement of the resulting 3-Phosphoglycerate results in the generation of a second ATP during glycolysis. The second method of ATP generation in our cells is called oxidative phosphorylation, which we will discuss in more detail in chapter 10. In this mechanism ADP and phosphate come together in a confined space of the ATP synthase and with the help of critical residues in the close vicinity of ADP and magnesium ions (Metal ion catalysis) phosphate forms an
anhydride bond with ADP. This reaction is near equilibrium in the active site, but the ATP would never be able to leave the site without a significant input of mechanical energy, that actively extrudes the ATP from its binding site, thereby pushing the reaction towards the formation of ATP instead of its hydrolysis, which is the favoured reaction to occur. Watch the video below to see ATP synthesis in action.

It seems a bit odd to call the second mechanism oxidative phosphorylation, when we saw that substrate level phosphorylation was also initiated with energy from an oxidation reaction. The names were developed from historical concepts. Oxidative phosphorylation is tightly coupled to the respiration inside the mitochondria and is therefore called oxidative. ATP production during glycolysis can occur in the absence of oxygen (fermentation), but the oxidation step still occurs and needs to be balanced by a reduction step resulting in the formation of lactate.

Now that we have generated ATP, we need to have a look how it is used to drive energy demanding reactions. If you want to understand the chemistry of ATP hydrolysis better, watch the video below. 

An important role of ATP is to generate mechanical energy in muscle. Here ATP is used to dislodge the motor protein myosin from the structural protein actin and to generate tension, which allows the power stroke to follow. See the video below.

Another important function of ATP is the establishment of ion gradients, which are critical for neural and muscle function. Watch the video below to see how ATP is used to pump ions across the membrane

In this book we are particularly interested in understanding metabolic processes. Synthesis of glutamine is an important step in the fixation of toxic ammonia, which is generated during the breakdown of amino acids. However, the amidation of glutamate to form glutamine is energetically unfavourable. With the help of ATP, we can make this reaction proceeding towards the formation of glutamine (Fig. 3).

Fig. 3 Involvement of ATP to synthesise glutamine

ATP is first used to form -glutamyl-phosphate. This intermediate can now be attacked by the free electron pair of ammonia and phosphate can leave after rearrangement of the electrons. Ammonia could condensate with gamma-glutamyl-phosphate due to phosphate acting as a leaving group. These examples illustrate how ATP is used to provide energy for many reactions occurring in our body.

ATP is very useful to energise condensation and group transfer reactions and also to induce conformational changes in proteins. However, synthesis of many biomolecules requires reduction of the molecule. One of the most frequently used reactions in our body is the reduction of a keto-group to a hydroxyl-group, a reaction used to synthesise fatty acids and also to generate lactate from pyruvate. ATP is not of much help here, because no change of the oxidation state occurs during ATP hydrolysis. For these reactions we use NADPH or NADH. The key element of both molecules is the nicotinamide ring attached to ribose (Fig. 1). The nicotinamide moiety has a permanent positively charged nitrogen atom that acts as an electron sink, allowing the transfer of two electrons. The extra negative charge is accommodated by reducing one of the double bonds of the nicotinamide ring and adding a proton. The reduced nicotinamide is stable and can store two electrons plus the associated proton until they are released when the NADH moves to another enzyme reducing another molecule.

NADH and NADPH carry out the same reaction, namely the transfer of two electrons and a proton. NADH is generated in catabolic processes, transferring the electrons to the mitochondria for respiration (Chapter 9), NADPH is used to transfer electrons to molecules that are newly synthesised (anabolic processes). Remember: Most anabolic processes use NADPH, catabolic processes use NADH. Think about the following two questions:

Let us have a look at reactions that require the use of NADH or NADPH. At this point we are not going into the detail why NADH or NADPH is used in the reactions. However, it is clear that reduction of these molecules occurs through the donation of two electrons and two protons.

Fig. 4 Examples of redox reactions in biological systems

As we saw earlier, oxidation reactions are generally favourable, thus reduction reactions require the input of NADPH or NADH to proceed from reactants to substrates as indicated in Fig. 4. All of these reactions are redox reactions, because the substrate becomes reduced while NADPH or NADH are oxidised. The major pathway to recharge NADPH is the Pentose-Phosphate Pathway (Chapter 17), the major pathway to generate NADH inside mitochondria is the citric acid cycle (Chapter 9), in the cytosol it is glycolysis Chapter 7).

The capability to donate or accept electrons is known as the redox potential. It is defined against the standard of protons accepting electrons thereby becoming hydrogen gas. Like free energy, the redox potential is defined under standard conditions and the real condition inside the cell may be quite different. There is a strict relationship between the free energy of a redox reaction and its redox potential: △G0=−zF△E0 In this equation z is the number of electrons shifted, F is the Faraday constant and E0 is the standard redox potential. In Table 1 a negative redox potential shows a propensity to donate electrons (i.e. go from product to substrate), while a positive redox potential indicates a reaction that prefers to accept electrons (i.e. the reduction of oxygen to form water). These reactions are called half-reactions because the shown reaction donates electrons, and another reaction is required to accept them. Electrons move spontaneously from more negative to more positive half reactions. For example, NADPH can reduce GSSG (two glutathione molecules linked by a disulfide bridge) into two molecules of glutathione.

Substrate Product Redox Potential
Acetyl-CoA +2e- +2H+
Pyruvate
-0.48V
NAD(P)+ + 2e- + 2H+
NAD(P)H + H+
-0.32V
Glutathione +2e- + 2H+
GS-SG
-0.23V
FAD + 2e- +2H+
FADH2
-0.22V
Pyruvate +2e- +2H+
Lactate
-0.18V
Oxaloacetate + 2e- + 2H+
Malate
-0.17V
2H+ +2e-
H2
0.00V
Cytochrome c (Fe3+) +1e-
Cytochrome c (Fe2+)
+0.23V
0.5 O2 + 2e- + 2H+
H2O
+0.82V

At this point we do not understand all the reactions in Table 1, but these will be explained later in the book. However, we observe that nutrient-derived metabolites typically have a negative redox potential, while iron complexes (cytochrome) and oxygen have positive redox potentials. You will see later that oxygen and iron complexes are intensively used to accept electrons in biological reactions. 

 

The standard redox potential assumes a ratio of 1:1 between substrates and products. Similar to ATP, living cells like to keep certain reactions away from equilibrium. NADPH is mostly reduced, while NAD is mostly oxidised. This reflects their biological role. NADPH reduces metabolic intermediates to form reduced biomolecules, such as fatty acids. NADH brings electrons to the mitochondria where they are used to generate water.

 

It is important to realise that in comparison to the amounts of macronutrients we metabolise, the amounts of NAD/NADH and ATP/ADP are relatively small. This leads automatically to the very important principle of metabolic coupling.

 
Fig. 5: Recycling of cofactors regulates metabolic pathways through demand.

Any metabolic pathway that produces ATP will quickly come to a halt because of the lack of ADP available for phosphoryl transfer. Metabolic pathways are best viewed as supply and demand chains. If there is a high demand for ATP, the pathway will run fast, if there is less demand metabolism will slow down. The same is true for pathways generating NADH. They come to halt in the absence of respiration. NADH needs to deliver electrons for the generation of water, which reforms NAD+. 

When you exercise, your heart starts pumping blood much faster than at rest. This provides more oxygen and glucose to muscles, where respiration and ATP use increases. This is metabolic coupling at work.

So far you have learned about ATP and NAD(P)H and their role in storing and transferring energy. You need to understand one more principle how to facilitate biological reactions. Many biological molecules are not overly reactive. If they were, our metabolism would be littered with unwanted reactions. We will see that enzymes facilitate biological reactions by reducing their activation energy. However, enzymes cannot change the equilibrium of a reaction. It would be helpful to have means to activate metabolic intermediates to make them more reactive. You have already seen an example when ATP was used to generate a phosphorylated intermediate (gamma-glutamyl-phosphate), which was more reactive than the original metabolite. Carboxyl-groups are found in many metabolites but tend to have little reactivity, because of the delocalisation of the electron between both oxygens.  A frequently used molecule to activate carboxyl-groups is Coenzyme A (Fig. 5). 

Fig. 5: Coenzyme A attached to acetate (blue).

You may notice some similarity to ADP, which is underlying a third of the molecule on the right. However, this is once again only relevant for the attachment to the enzyme using this molecule. The important part of the molecule is a sulfhydryl group (-SH) at the left end, which in Fig. 5 forms a thioester with acetate. The whole molecule is called Acetyl-CoA and is arguably the most central metabolite in biochemistry. Acetyl-CoA is the final degradation product of carbohydrates, fats and many amino acids, before they are fully oxidised to carbon dioxide. It is also the starting point to synthesize fatty acids, cholesterol and ketone bodies.

It is revealing that thioesters are obligatory intermediates in several key processes in which ATP is either used or regenerated. Thioesters are involved in the synthesis of all esters, including those found in complex lipids. They also participate in the synthesis of a number of other cellular components, including peptides, fatty acids, sterols, terpenes, porphyrins, and others. In addition, thioesters are formed as key intermediates in several particularly ancient processes that result in the assembly of ATP. In both these instances, the thioester is closer than ATP to the process that uses or yields energy. In other words, thioesters could have actually played the role of ATP in a "thioester world" initially devoid of ATP. Eventually, [these] thioesters could have served to usher in ATP through its ability to support the formation of bonds between phosphate groups.
Christian de Duve
(1995). "The Beginnings of Life on Earth". American Scientist. 83(5): 428–437. Bibcode:1995AmSci..83..428M.

What is so special about thioesters? In contrast to oxygen esters, electrons are less delocalised in a thioester bond, and therefore the bond is more reactive (Fig. 6).

Fig. 6: Lack of resonance stabilisation of electrons in a thioester results in higher free energy of hydrolysis.

Due to the enhanced reactivity, thioesters of carboxyl-group containing organic molecules can facilitate ester formation and condensation reactions. Because they are high-energy compounds, synthesis of thioesters requires ATP input.  Two examples are shown in Fig. 7. In panel (A) coenzyme A makes the methyl group of acetate nucleophilic. In panel (B) the free electron pair of the sulfhydryl-group acts as the nucleophile and AMP is the leaving group.

Fig. 7 Examples for the use of thioesters in metabolism.

Figure 7 illustrates several important principles of nutrient metabolism. Panel A shows a condensation reaction between acetyl-CoA and oxaloactetate. This reaction initiates the citric acid cycle (Chapter 9) and would not be possible using acetate as a reactant, because it is chemically too inert. Acetyl-CoA by contrast can form a reactive carbanion, which tautomerises into an enol conformation. As a result, the beta-carbon of acetyl-CoA has a much higher reactivity than that of acetate.  It is the free energy of the hydrolysis of the thioester that drives the citrate synthase reaction.  Panel B illustrates that the generation of a thioester requires ATP, consistent with its high energy of hydrolysis. Please note that instead of a phosphorylated intermediate, we are transferring AMP, which is used as a leaving group in the next reaction. The main reason for this type of activation by ATP is the extra energy gained from the hydrolysis of pyrophosphate (-33 kJ/mol). This drives the reaction towards the products. 

  • Unfavorable condensation reactions, such as phosphoryl-transfer, amidation reactions etc. can be energised with the help of ATP.
  • The energy of life is maintained by keeping the ATP hydrolysis reaction out of equilibrium.
  • Redox reactions can be energised by coupling an electron transfer reaction with a negative redox potential to one with a more positive redox potential.
  • NADPH is used for anabolic reactions, NADH (with a few exceptions) is used to transfer electrons to the respiratory chain.
  • ADP and NAD must be recycled when used in pathways that generate ATP and NADH. This supply and demand system is called metabolic coupling. 
  • Coenzyme A (CoA) and Adenosine-monophosphate (AMP) are used to activate organic molecules. The hydrolysis of the thioester formed between an organic acid and CoA can be used to drive a reaction. In the case of AMP transfer, the release of pyrophosphate and its subsequent hydrolysis can drive reactions.
  1. Fig. 1 By the author using ChemDraw
  2. Fig. 2 By the author using ChemDraw
  3. Fig. 3 Molecules2014, 19(9), 13161-13176; doi:10.3390/molecules190913161  open access article distributed under the Creative Commons Attribution License (CC BY 3.0) 
  4. Fig. 4 By the author using ChemDraw
  5. User:Bryan Derksen (original) and DMacks (talk) (color-change) [Public domain], via Wikimedia Commons
  6. Fig. 6 By the author using ChemDraw
  7. Fig. 7 By the author using ChemDraw