Energy Production and Storage for Life

Abstract

This will definitely be the most “chemical” chapter of the entire book. While we are interested in describing the physics of living organisms, one cannot escape the fact that, at the most microscopic level, a variety of molecules and vastly complex chemical reactions constitute the basis of all life processes. Understanding some basic principles of how energy is obtained and stored by the cells in what constitutes the vast book of metabolism is very helpful, to understand how this energy is then transported and used, turned into work and heat, for all the functions of the body. It is just amazing to realise how deeply rooted are all such chemical mechanisms: the fact that we can observe the same chemical synthesis pathways in such distant organisms as a bacterium, an oak tree, and a giraffe, tells that these fundamentals were already well established in the early days of the evolutionary path of life on Earth.

Appendices

Appendix C: The Molecules of Life

Water, ions, and a quantity small organic molecules, such as sugars, vitamins, fatty acids, account for about 80 % of living matter by weight. Of these small molecules, water is by far the most abundant. The remaining 20 % consists of macromolecules: proteins, polysaccharides, and nucleic acids. In Chap. 3 the attention was focused on these latter. In Chap. 4, ATP and ADP have taken the stage, together with a number of important enzymes among which FADH$_2$ and NADH. Notably, the nucleotides making up the nucleic acids DNA and RNA (see Appendix B) share the same basic chemical structure of ATP and ADP, and the coenzymes have chemical structures strictly derived from these same ones. Phospholipids and the multi-layered membranes they can form will be an important part of Chap. 5. And Chaps. 6–10 will be dominated by a quantity of highly specialised proteins.

Fig. 4.12

Chemical structure of nucleic acids. A nucleoside plus one, two or three phosphates makes a nucleotide. DNA and RNA are distinguished by the different bases attached to the central ribose by the glycosidic bond

The chemical structure of the nucleoside is shown in yellow, in Fig. 4.12: it is composed by a pentagonal sugar ring, the ribose, to which one of the five possible nucleosidic bases (in blue: A, G, T, C for DNA, and U replacing T for RNA) are attached by a glycosidic bond. The chemical difference between DNA and RNA is visible in the carbon atom labelled $2'$: DNA has a H atom, while RNA has an hydroxyl (OH) group. The carbon $3'$, where another OH is attached in both nucleic acids, is the site where a link with an adjacent base along the chain can be formed. Both DNA and RNA are composed by joining together nucleotides (a nucleoside plus its lateral phosphate chain) in the monophosphate form, i.e. carrying only one PO$_3^-$ side group (red in the Figure). In the monophosphate, one of the oxygen atoms is doubly bound to the phosphorus, as P = O, a second one is saturated by a hydrogen, as OH, and the last one is unsaturated and therefore negatively charged. When the next base is attached to either a DNA or RNA chain at the $5'$ end, the OH from one base reacts with the H at the $3'$ position of the other, and the two form a H$_2$O molecule while the two bases are covalently bonded together. This is called a phosphodiester bond, the newly formed PO$_4^{3-}$ group being overall negatively charged. Such a bond can be broken by adding back the water molecule, in a process of hydrolysis. Because of the presence of the OH in the $2'$ position, hydrolysis of the -O-P-O- phosphodiester bond is much easier (energetically less costly) in RNA than in DNA. This is one reason for the higher catalytic activity of RNA.

Fig. 4.13

The ATP (above, left) and ADP (right) nucleotides. Each contains adenine as the base, and respectively three or two phosphate groups, which are charged in the former, and neutralised by H in the latter. The coenzymes FAD (middle, left), NAD$^+$ (right), and acetyl-CoA (below). Note the similarity in the chemical structure, built from an adenine base and two phosphate groups (like in ATP and ADP), and a second moiety (riboflavine in FAD, nicotinamide in NAD, mercapto-ethylamine in the acetyl-CoA) linked by a phosphodiester bond to the phosphates

As shown in Fig. 4.12, nucleotides can also occur in the diphosphate and triphosphate form, with two or three PO$_4^{3-}$ groups consecutively attached to the $5'$ carbon atom. This structure is the same found in the ATP and ADP. Figure 4.13 (top row) shows the structure of these molecules, which have one adenine base attached to the ribose and, respectively, three or two phosphate groups (their names, adenosin-tri-phosphate, and adenosin-di-phosphate, specify exactly this feature). It should be noted that in their ’naked’ form ATP and ADP have a large negative charge of -4 and -3 respectively. Despite the coordination with water molecules, such large charges are difficult to stabilise, therefore in biologically-relevant conditions these species are always complexed, typically with Mg$^{2+}$ ions, to [ATP-Mg]$^{2-}$ and [ADP-Mg]$^-$. Also the other bases (G, T, C) can form di- or tri-phosphates, however these species will be rarely encountered in the subjects discussed here, with the possible exception of GTP, guanosine triphosphate.

A subset of important players is represented by the coenzymes. Flavin adenine dinucleotide, or FADH$_2$, is a redox cofactor that is created during the Krebs cycle and utilised during the last part of respiration, the electron transport chain. Nicotinamide adenine dinucleotide, or NADH, is a similar compound actively used as well in the electron transport chain. In Fig. 4.13 (middle row) the chemical structures of FAD and NAD$^+$ are shown. In the former, the two N can be reduced to NH, thus giving FADH$_2$; in the latter, the 4$^{\prime }$ CH can be reduced to CH$_2$, thus giving NADH. As we learned in this chapter, both species are oxidised back to their original state, in the parallel reduction of cytochrome. Both molecules are based on an adenine nucleotide (ATP), linked to another moiety by a phosphodiester bond between two PO$_4^{3-}$ groups, which are partly saturated in FADH$_2$. The moiety in the latter is a riboflavin, while it is a nicotinamide linked to a sugar ribose in NADH.

Acetyl-coenzyme-A or acetyl-CoA (Fig. 4.13, bottom row) is produced during the breakdown of carbohydrates through glycolysis, as well as by the beta-oxidation of fatty acids. This fundamental coenzyme feeds the two carbon atoms of its end-group acetyl (-COOH), into the Krebs cycle, which will be oxidised to CO$_2$ and water, to produce energy stored in ATP. The terminal acetyl group is linked by a strong bond to the S atom of mercapto-ethylamine; hydrolysis of this bond is exoergic (it releases a $\varDelta G=-31.5$ kJ). It is this bond that makes acetyl-CoA one of the “high energy” compounds. Overall, about 11 ATP and 1 GTP molecules are obtained per acetyl group that enters the Krebs cycle.

Note that all these coenzymes have a similar structure shared with ATP and ADP, in that one ADP moiety is common to all of them.

The triglycerides (also called triacylglycerols, TG, or triacylglycerides, TAG) are compound molecules in which the three hydroxyls (OH) of a glycerol are linked to three fatty acids. Such molecules are the main constituents of the vegetable oils and of animal fat. In Fig. 4.14, the chemical structure and general formula of triglycerides is given. Here R$_1$, R$_2$ and R$_3$ are three, generally non-identical fatty acids, with general formula (-CH$_2$)$_n$-COOH, and length ranging from $n=4$ to 22 carbon atoms. However, a length between 16 and 18 is the most commonly observed. Shorter carbon chains are observed in the butyric acid, the principal component of home butter. The glycerol is a polyol, familiarly know as glycerine, usually produced as side-product in the glycolysis. Liver and adipose tissue can supply glycerol when glycolysis is scarce, by using an alternate metabolic path involving amino acids.

Fig. 4.14

Stereochemical formula of a triglycerid. The central glycerol (in the green box) is attached to three lipid chains, (CH$_2$)$_n$, indicated as R$_{1,2,3}$. In the schematic on the left, each vertex $\diagup \diagdown$ or $\diagdown \diagup$ implicitly indicates a CH$_2$ group

Practically all naturally occurring fatty acids have an even number of carbon atoms, because they are all bio-synthesised starting from acetic acid, the smallest carboxylic acid with chemical formula CH$_3$COOH. The carbon chains in triglycerides can be saturated or unsaturated, indexsaturated/unsaturated, fatty chain i.e., they can contain one or more double carbon bonds C=C, in each chain (see also Appendix D, about the chemical nature of phospholipids).

Most natural fats, such as butter, lard, tallow, are made from a complex mixture of triglycerides. Due to this, they melt progressively over a wide interval of temperatures. Cocoa butter is atypical, since it is made of only one type of triglyceride, in which the three chains are a palmitic, an oleic and a stearic acid, and has therefore a well-defined melting point. This is likely the reason why chocolate melts in the mouth without giving off a too “fatty” feeling.

Carbohydrates are a widely diverse group of compounds that are ubiquitous in nature. More than 75 % of the dry weight of the plant world is carbohydrate in nature, particularly cellulose, hemicellulose and lignin. Among carbohydrates, sugars occupy a special position, due to their variety of structures and bonding, allowing a chemical flexibility vastly superior even to proteins, with combinations ranging from simple monosaccharides to polymers made of millions of units (Fig. 4.15).

Fig. 4.15

Different representations of the five-carbon sugar ribose. a, b Fischer projection of D-ribose and L-ribose. In this representation it is evident the enantiomerism, the two molecules being mirror images of each other. c, d Furanose and e, f pyranose molecular structure of D-ribose and 2-deoxy-D-ribose. These two sugars link in the furanose form to phosphate groups (via C3 and C5) to build up the backbone of the RNA or DNA chain, and provide the linkage (via C1) between the backbone and the nucleobases

Monosaccharides are linear or ring-shaped molecules with four to seven carbon atoms. Because these molecules have multiple asymmetric carbons, they can exist as isomers that are not mirror images of each other (enantiomers), indicated by the symbols D and L. Among the most important sugars to be found in the cell environment, we find the five-carbon D-ribose that is at the heart of RNA, and of DNA in the deoxyribose form (one O is lost from the OH group in the 1$^{\prime }$ carbon); and the six-carbon D-glucose, produced in the photosynthesis and at the centre of the glycolysis cycle (note that L-ribose does not exist in nature, and also L-glucose is rarely found). Such sugars in solution are nearly always in the closed ring form, with only $\sim$0.1–0.5 % of the molecules in the open-chain structure. When the chain closes into a ring, a pentagonal (furanose) or hexagonal (pyranose) structure can be formed, by excluding one of the carbons from the ring. The hexagonal structure is more common in ribose, and almost exclusive in glucose. However, the ribose making up the structure of DNA and RNA is always in the pentagonal form.

Polysaccharides or glycans are formed by joining together any combination of monosaccharides, via a glycosidic bond (the same name is given also to the bond between a sugar and any other molecule). They range in structure from linear to highly branched. Examples include storage polysaccharides such as starch and glycogen, and structural polysaccharides such as cellulose and chitin. A dense layer of glycans is found on the outer surface of many cells, the glycocalyx. Glycans can combine with proteins in various forms, giving rise to peptidoglycans. The outer surface of bacterial cells is covered by a cortex of peptidoglycans arranged in a nearly crystalline form, providing a kind of exoskeleton that gives the bacterium structural strength and resistance against osmotic pressure.

Proteins are the other majority component of cells. The assembly of proteins from a sequence of amino acids translated by the mRNA was briefly described in the Appendix B. Once linked by peptide bonds in the primary structure, the long sequence of amino acids must however get folded into a three-dimensional structure, for the protein to become fully functional.

Starting from the primary structure, a variety of local interactions (van der Waals and electrostatic interactions between charges and dipoles, $\pi$-stacking, hydrogen bonds, hydrophobic effect) make up contacts between different portions of the peptide chain, and make it fold and bend into the secondary structure. The properties of the amino acids are listed in Fig. 3.14 of Appendix B. According to their degree of hydrophobicity, parts of the structure of the protein can adjust to minimise contact with water. Although it is impossible to provide an exhaustive list of the thousands different proteins necessary for the functioning of living organisms, there are some structural motifs in the secondary structure, which are recurrently found in the architecture of diverse proteins. Such “universal” motifs allow to recognise and classify common functional substructures, even in proteins performing completely different functions. The most important such motifs are the alpha helix and the beta sheet, shown in Fig. 4.16(a, b). The alpha helix gets its name from the central C atom of each amino acid, called the “alpha” carbon. In some regions of the protein, neighbouring N-H and C=O groups can form hydrogen bonds, which make for a more solid bonding than provided by the longer-range Van der Waals and electrostatic forces. A locally helical structure can arise from such bonds, every fourth $\alpha$-C being found on top of each other, with a typical helix period of 0.54 nm (compare to 0.34 nm in DNA). The $\beta$-sheet is also formed by the same type of hydrogen bonds between N-H and C=O, but in this case the repeated structure is a multiply-folded flat pattern of roughly aligned strips of amino acids. The alignment of the strips can be parallel or antiparallel, according to the arrangement of the residues, either facing or opposing each other on each side of the strip. Such easily recognisable structures as the $\alpha$-helix and the $\beta$-sheet are ubiquitous in all proteins, and are usually represented by a helical ribbon, and by a flat arrow, respectively, in the ternary structure, the actual 3-D form of the protein (Fig. 4.16c).

Fig. 4.16

Schematic of a $\alpha$-helix and b $\beta$-sheet subunits of protein secondary structure. c Tertiary 3-D structure of the protein Src-kinase, drawn in the “cartoon” style, to highlight the presence of $\alpha$-helices (purple) and $\beta$-sheets (yellow). The strings drawn in grey correspond to subunits with undefined (random) structure

Any protein can be composed by several different domains, or subunits that can fold independently into the 3-D structure. Some proteins can be composed by several repeats of one same domain. For example titin, the largest protein found in the human body, whose sequence of about 27,000 amino acids is made for about 90 % of two modules, the Ig (immunoglobulin) and the FN3. Note that these same domains are observed, with some variants, also in several other proteins.

When the protein functions as enzyme, its structure hosts one (rarely more than one) active site. These may appear as “pockets” or “holes” in the tertiary structure, in which a small organic molecule (the ligand) can fit and be temporarily bound (for example, myosin binding ATP to perform the power stroke in muscle contraction, see Chap. 9). The portion of the active site where the ligand binds is the binding site. The “lock-and-key” model of the ligand-enzyme interaction predicts that there is a perfect geometrical fitting between the two, such that the binding does not induce any further structural change in the couple. In the “induced-fit” model, the active site is modified by the entry of the ligand, and returns to its unperturbed shape when the ligand is released.

Problems

4.1

The $\varDelta G$ of metabolic reactions

Consider a typical metabolic reaction in the form A$\rightarrow$B. Its standard free energy change is 7.5 k Jmol$^{-1}$.

• (a) Calculate the equilibrium constant for the reaction at 25 $^{\circ }$C.

• (b) Calculate the $\varDelta G$ at 37 $^{\circ }$C, when the concentration of A is 0.5 mM and the concentration of B is 0.1 mM. Is the reaction spontaneous?

• (c) Under which conditions might the reaction proceed in the cell?

4.2

Switching from ATP to ADP

Adenylate kinase (ADK) is a phosphotransferase enzyme that catalyses the interconversion of adenine nucleotides, and plays an important role in maintaining the right concentrations of ATP and ADP in the cell (“cellular energy homeostasis”). The reaction can be schematised as:

$$\begin{aligned} \text {ATP} + \text {AMP} \leftrightarrows 2 \,\, \text {ADP} \end{aligned}$$

Given the concentrations of [ATP] = 5 mM, [ADP] = 0.5 mM, calculate the $[\text {AMP}]$ concentration at pH = 7 and 25 $^{\circ }$C, under the condition that the adenylate kinase reaction is at equilibrium.

4.3

Energy harvesting

Calculate the absolute yield of ATP per mole, when a substrate is completely oxidised to CO$_2$, in the case of:

(a) pyruvate (CH$_3$-CO-COO$^-$),

(b) lactate (CH$_3$-CH-OH-CO$_2^-$),

(c) glucose (C$_6$H$_{12}$O$_{6}$),

(d) fructose 1,6-diphosphate (C$_6$H$_{14}$O$_6$(PO$_3^{2-}$)$_2$).

4.4

Human blood

The kidneys help control the amount of phosphate in the blood. Extra phosphate is filtered by the kidneys and passes out of the body in the urine. A high level of phosphate in the blood is usually caused by a kidney problem. Normal levels of potassium in human blood should be in the range 3.5–5 mM. However, the phosphate ion can be found in any of its protonation states, H$_3$PO$_4$ (neutral), H$_2$PO$^-_4$, HPO$^{2-}_3$ and PO$^{3-}_4$ (this is the mix called inorganic phosphate, indicated with Pi, see also footnote to p. 114). Given the equilibrium constants for the three reactions:

$$\begin{aligned} \text {H}_3\text {PO}_4 + \text {H}_2\text {O}&\leftrightarrow \text {H}_3\text {O}^+ + \text {H}_2\text {PO}^{-}_4 \\ \text {H}_2\text {PO}^{-}_4 + \text {H}_2\text {O}&\leftrightarrow \text {H}_3\text {O}^+ + \text {H}\text {PO}^{2-}_4 \\ \text {H}\text {PO}^{2-}_4 + \text {H}_2\text {O}&\leftrightarrow \text {H}_3\text {O}^+ + \text {PO}^{3-}_4 \\ \end{aligned}$$

respectively equal to $K_1=7.5\times 10^{-3}$, $K_2=6.2\times 10^{-8}$, $K_3=2.2\times 10^{-13}$, calculate the relative concentrations of the different phosphate ions in human blood at physiologic pH = 7.4.

4.5

Gym doesn’t slim

Fats are usually metabolised into acetyl-CoA and then further processed through the citric acid (Krebs’) cycle. However, glucose also could be synthesised from oxaloacetate, one of the intermediates during the citric acid cycle. Why, then, after some hours of exercise depleted our carbohydrate reserve, do we need to replenish those stores by eating again carbohydrates? Why do we not simply replace them, by converting some stored fats into carbohydrates?

(Hint: look at the number of carbon atoms entering and exiting the Krebs’ cycle)

4.6

Pigeon muscles love citrate

The activity of the citric acid cycle can be monitored by measuring the amount of O$_2$ consumed. The greater the rate of O$_2$ consumption, the faster the rate of the cycle, the faster the rate of ATP production. Hans Krebs in 1937 used this type of experiments, working with fragments of pigeon breast muscle, very rich in mitochondria. In one set of experiments, he measured O$_2$ consumption in the presence of carbohydrate only, and in the presence of carbohydrate plus 3 $\upmu$mol of citrate (C$_6$H$_8$O$_7$). After 2h30 he measured a consumption of 49 $\upmu$M with glucose only, and 85 $\upmu$M when citrate was added. Complete oxidation of citrate follows this chemical equation:

$$\begin{aligned} \text {C}_6\text {H}_8\text {O}_7 + x \,\, \text {O}_2 \rightarrow y \,\, \text {CO}_2 + z \,\, \text {H}_2\text {O} \end{aligned}$$

• (a) What is x, y and z, and how many moles of oxygen are consumed in the experiment after adding the citrate?

• (b) Given the experimental result, what implications does this have for metabolism?

4.7

Antibiotics

Oligomycin-A is a natural antibiotic, isolated from the Streptomyces bacteria, which works by inhibiting the action of the ATP-synthase pump. It is sometimes used in laboratory research about ion channels, but it is never adopted in any pharmaceutical prescription drugs, because it is highly toxic. What is the main reason for it being so dangerous for animals?

4.8

Transmembrane proteins

Transmembrane proteins are quite big molecules that cross the cell membrane, exposing part of the structure both to the inside and the outside of the cell. By looking at the amino acid sequence of one such proteins, it is seen that it includes four regions characterised by strongly hydrophilic amino acids, separated by regions containing mostly hydrophobic amino acids. Draw a sketch of the tertiary structure arrangement across the membrane.

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