Solved Examples

How to Use This Section

This chapter is examined in a very particular way. Almost every question is "name the compound", "give the number", "state the one function", "which of these is not", or a short comparison between two things that sound alike. There is almost nothing to derive and nothing to calculate - the marks sit in the exact names, the exact numbers and the one line that separates two look-alike terms.

That makes this chapter one of the easiest to score full marks in, and one of the easiest to lose marks in through a single swapped word. The filtrate is the acid-soluble pool and the retentate is the acid-insoluble fraction. A nucleoside is base plus sugar and a nucleotide is base plus sugar plus phosphate. Adenine and guanine are the purines and everything else is a pyrimidine. Collagen is the most abundant protein in the animal world and RuBisCO in the whole of the biosphere. Each of those pairs has cost more marks than any reasoning question in the chapter.

Work through the three tiers in order.

1. Tier 1 - Concept Checks. One fact per question, straight from the text. If you cannot answer these without stopping, go back to the section they came from before moving on.
2. Tier 2 - Application and Identification. A compound is described, or a small situation is given, and you name it and justify the name. This is exactly how the "identify the biomolecule" question is set.
3. Tier 3 - Comparisons and Long Answers. The two-column differences and the joined-up descriptions - micromolecule against macromolecule, DNA against RNA, the four levels of protein structure, and the six classes of enzymes.

The eleven chapter-end exercises are all answered somewhere in this chapter. The last block lists where each one is answered and works out the three that are not covered anywhere else.

The Facts These Questions Draw On

Grinding a tissue. Grind any living tissue in trichloroacetic acid, $\mathrm{Cl_3CCOOH}$, with a mortar and a pestle and strain the thick slurry through cheesecloth or cotton. The filtrate is the acid-soluble pool and holds thousands of organic compounds, the small ones. The retentate is the acid-insoluble fraction and holds proteins, nucleic acids, polysaccharides and lipids. To identify one compound you extract, separate, isolate and purify it, then analytical techniques give the molecular formula and the probable structure. All the carbon compounds that we get from living tissues can be called biomolecules.

Wet weight, dry weight and ash. Weigh a fresh living tissue - that is the wet weight. Dry it, all the water evaporates, and what remains gives the dry weight. Burn it fully, all the carbon compounds are oxidised to $\mathrm{CO_2}$ and water vapour and are removed, and what is left is called ash. The ash contains inorganic elements like calcium and magnesium. Inorganic compounds like sulphate and phosphate are also seen in the acid-soluble fraction. Elemental analysis gives the elemental composition; analysis for compounds gives the organic and inorganic constituents.

The elements. The same elements present in a sample of the earth's crust are also present in a sample of living tissue, but the relative abundance of carbon and hydrogen is much higher in a living organism than in the earth's crust. Hydrogen 0.14 in the crust against 9.5 in the human body; carbon 0.03 against 18.5; oxygen 46.6 against 65.0; nitrogen very little against 3.3; sulphur 0.03 against 0.3; sodium 2.8 against 0.2; calcium 3.6 against 1.5; magnesium 2.1 against 0.1; silicon 27.7 against negligible. Oxygen is the most abundant element in the human body at 65.0 per cent, but water is the most abundant chemical in living organisms. The inorganic constituents are $\mathrm{Na^+}$, $\mathrm{K^+}$, $\mathrm{Ca^{2+}}$, $\mathrm{Mg^{2+}}$, $\mathrm{H_2O}$ and compounds such as $\mathrm{NaCl}$, $\mathrm{CaCO_3}$, $\mathrm{PO_4^{3-}}$ and $\mathrm{SO_4^{2-}}$.

Amino acids. Amino acids are organic compounds containing an amino group and an acidic group as substituents on the same carbon, the $\alpha$-carbon, hence they are called $\alpha$-amino acids. They are substituted methanes, with four substituent groups occupying the four valency positions - hydrogen, the carboxyl group $\mathrm{-COOH}$, the amino group $\mathrm{-NH_2}$, and a variable group designated as the R group. Based on the nature of the R group there are many amino acids, but those which occur in proteins are only of twenty types. R group hydrogen gives glycine, a methyl group gives alanine, hydroxy methyl gives serine. The chemical and physical properties of amino acids are essentially those of the amino, carboxyl and R functional groups. By the number of amino and carboxyl groups they are acidic - glutamic acid, basic - lysine, or neutral - valine. Tyrosine, phenylalanine and tryptophan are the aromatic amino acids. Non-essential amino acids are those our body can make; essential amino acids we get through our diet or food, so dietary proteins are the source of essential amino acids.

The zwitterion. A particular property of amino acids is the ionizable nature of the $\mathrm{-NH_2}$ and $\mathrm{-COOH}$ groups, so the structure of amino acids changes in solutions of different pH. In a strongly acidic solution the amino group takes up a proton and the molecule is net positive. In a strongly basic solution the carboxyl group gives up a proton and the molecule is net negative. At an intermediate pH the $\mathrm{-COOH}$ loses a proton and the $\mathrm{-NH_2}$ gains one, so the molecule carries a positive and a negative charge on the same molecule while being electrically neutral overall - this is the zwitterionic form.

Lipids and fatty acids. Lipids are generally water insoluble. A fatty acid has a carboxyl group $\mathrm{-COOH}$ attached to an R group, and the R group could be a methyl $\mathrm{-CH_3}$, an ethyl $\mathrm{-C_2H_5}$, or a higher number of $\mathrm{-CH_2}$ groups, from 1 carbon to 19 carbons. Palmitic acid has 16 carbons including the carboxyl carbon. Arachidonic acid has 20 carbon atoms including the carboxyl carbon. Fatty acids are saturated, without a double bond, or unsaturated, with one or more $\mathrm{C=C}$ double bonds.

Glycerol and the glycerides. Another simple lipid is glycerol, which is trihydroxy propane. Many lipids have both glycerol and fatty acids, where the fatty acids are found esterified with glycerol, giving monoglycerides with one fatty acid, diglycerides with two and triglycerides with three. These are also called fats and oils based on melting point - oils have a lower melting point, for example gingelly oil, and hence remain as oil in winters. Fats and oils are glycerides in which fatty acids are esterified to glycerol. Some lipids have phosphorous and a phosphorylated organic compound in them; these are phospholipids, they are found in the cell membrane, and lecithin is one example. Phospholipids contain, in addition to glycerol and fatty acids, a phosphorylated nitrogenous compound. Some tissues, especially the neural tissues, have lipids with more complex structures.

Nitrogen bases, nucleosides and nucleotides. Living organisms have a number of carbon compounds in which heterocyclic rings can be found, and some of these are the nitrogen bases - adenine, guanine, cytosine, uracil and thymine. Adenine and guanine are substituted purines; cytosine, uracil and thymine are substituted pyrimidines, and the skeletal heterocyclic rings themselves are called purine and pyrimidine respectively. When a nitrogen base is found attached to a sugar, it is called a nucleoside. If a phosphate group is also found esterified to the sugar, it is called a nucleotide. The nucleosides are adenosine, guanosine, thymidine, uridine and cytidine. The nucleotides are adenylic acid, thymidylic acid, guanylic acid, uridylic acid and cytidylic acid. Nucleic acids like DNA and RNA consist of nucleotides only, and DNA and RNA function as genetic material.

Metabolites. A list of biomolecules would run to thousands of organic compounds including amino acids and sugars, and we can call these biomolecules metabolites. In animal tissues one notices all such categories of compounds - amino acids, sugars, fatty acids, glycerol, nucleotides, nucleosides and nitrogen bases - and these are primary metabolites. Primary metabolites have identifiable functions and play known roles in normal physiological processes. When one analyses plant, fungal and microbial cells, one would see thousands of compounds other than these primary metabolites - alkaloids, flavonoids, rubber, essential oils, antibiotics, coloured pigments, scents, gums and spices - and these are called secondary metabolites. Pigments - carotenoids, anthocyanins. Alkaloids - morphine, codeine. Terpenoides - monoterpenes, diterpenes. Essential oils - lemon grass oil. Toxins - abrin, ricin. Lectins - concanavalin A. Drugs - vinblastin, curcumin. Polymeric substances - rubber, gums, cellulose. We do not at the moment understand the role or functions of all the secondary metabolites in host organisms, however many of them are useful to human welfare - rubber, drugs, spices, scents and pigments - and some have ecological importance.

The molecular weight divide. All the compounds found in the acid soluble pool have molecular weights ranging from 18 to around 800 daltons (Da) approximately. The acid insoluble fraction has only four types of organic compounds - proteins, nucleic acids, polysaccharides and lipids - and these classes, with the exception of lipids, have molecular weights in the range of ten thousand daltons and above. Biomolecules are therefore of two types: those with molecular weights less than one thousand dalton are usually referred to as micromolecules or simply biomolecules, and those found in the acid insoluble fraction are called macromolecules or biomacromolecules. The molecules in the insoluble fraction, with the exception of lipids, are polymeric substances.

The lipid caveat. Lipids are small molecular weight compounds whose molecular weights do not exceed 800 Da, yet they land in the acid insoluble fraction. The reason is that lipids are present not only as such but also arranged into structures like the cell membrane and other membranes. When we grind a tissue we disrupt the cell structure, the membranes are broken into pieces and form vesicles which are not water soluble, and therefore these membrane fragments in the form of vesicles get separated along with the acid insoluble pool. Lipids are thus not strictly macromolecules. The acid soluble pool represents roughly the cytoplasmic composition, the macromolecules from cytoplasm and organelles become the acid insoluble fraction, and together they represent the entire chemical composition of living tissues or organisms. Average composition of cells as per cent of total cellular mass - water 70 to 90, proteins 10 to 15, carbohydrates 3, lipids 2, nucleic acids 5 to 7, ions 1.

Proteins. Proteins are polypeptides - linear chains of amino acids linked by peptide bonds - and each protein is a polymer of amino acids. There are 20 types of amino acids, such as alanine, cysteine, proline, tryptophan and lysine, so a protein is a heteropolymer and not a homopolymer. A homopolymer has only one type of monomer repeating "n" number of times, the standard example being cellulose, which is built of glucose alone. Proteins carry out many functions - some transport nutrients across the cell membrane, some fight infectious organisms, some are hormones, some are enzymes. Collagen - intercellular ground substance. Trypsin - enzyme. Insulin - hormone. Antibody - fights infectious agents. Receptor - sensory reception of smell, taste and hormone. GLUT-4 - enables glucose transport into cells. Collagen is the most abundant protein in the animal world. Ribulose bisphosphate Carboxylase-Oxygenase (RuBisCO) is the most abundant protein in the whole of the biosphere.

Polysaccharides. The acid insoluble pellet also has polysaccharides (carbohydrates) as another class of macromolecules. Polysaccharides are long chains of sugars - threads containing different monosaccharides as building blocks. Cellulose is a polymeric polysaccharide consisting of only one type of monosaccharide, glucose, so it is a homopolymer. Starch is a variant of this, present as a store house of energy in plant tissues; animals have another variant called glycogen; inulin is a polymer of fructose. In a polysaccharide chain such as glycogen the right end is called the reducing end and the left end the non-reducing end, and it has branches. Starch forms helical secondary structures and can hold $\mathrm{I_2}$ molecules in the helical portion, and the starch-$\mathrm{I_2}$ complex is blue in colour. Cellulose does not contain complex helices and hence cannot hold $\mathrm{I_2}$. Plant cell walls are made of cellulose, and paper made from plant pulp and cotton fibre are cellulosic. More complex polysaccharides have as building blocks amino-sugars and chemically modified sugars such as glucosamine and N-acetyl galactosamine. Exoskeletons of arthropods have a complex polysaccharide called chitin, and these complex polysaccharides are mostly homopolymers.

Nucleic acids. The other type of macromolecule in the acid insoluble fraction of any living tissue is the nucleic acid, and these are polynucleotides. Together with polysaccharides and polypeptides, nucleic acids comprise the true macromolecular fraction of any living tissue or cell. For nucleic acids the building block is a nucleotide, and a nucleotide has three chemically distinct components - a heterocyclic compound, a monosaccharide, and a phosphoric acid or phosphate. Phosphoric acid is $\mathrm{H_3PO_4}$ and it sits in the chain as the phosphate group, $\mathrm{PO_4^{3-}}$. The heterocyclic compounds in nucleic acids are the nitrogenous bases - adenine, guanine, uracil, cytosine and thymine - and adenine and guanine are substituted purines while the rest are substituted pyrimidines. The sugar found in polynucleotides is either ribose, a monosaccharide pentose, or 2-deoxyribose. A nucleic acid containing deoxyribose is called deoxyribonucleic acid (DNA), while that which contains ribose is called ribonucleic acid (RNA). Nucleic acids serve as genetic material; they carry hereditary information and are passed on from parental generation to progeny.

The four levels of protein structure. Proteins are heteropolymers containing strings of amino acids, and biologists describe the protein structure at four levels - primary, secondary, tertiary and quaternary. Primary structure is the sequence of amino acids, that is the positional information in a protein - which is the first amino acid, which is second, and so on. A protein is imagined as a line, the left end represented by the first amino acid, which is also called the N-terminal amino acid, and the right end by the last, the C-terminal amino acid. A protein thread does not exist throughout as an extended rigid rod; the thread is folded in the form of a helix, similar to a revolving staircase, only some portions of the thread are arranged in the form of a helix, and other regions are folded into other forms - all of this is the secondary structure. In proteins, only right handed helices are observed. The named forms are the $\alpha$-helix and the $\beta$-pleated sheet. In addition the long protein chain is also folded upon itself like a hollow woolen ball, giving rise to the tertiary structure, which gives us a three dimensional view of a protein and is absolutely necessary for the many biological activities of proteins. Some proteins are an assembly of more than one polypeptide or subunits, and the manner in which these individual folded polypeptides or subunits are arranged with respect to each other - a linear string of spheres, or spheres arranged one upon each other in the form of a cube or plate - is the architecture of a protein, otherwise called the quaternary structure. Adult human haemoglobin consists of 4 subunits, two of which are identical to each other - two subunits of $\alpha$ type and two subunits of $\beta$ type together constitute the human haemoglobin, $\mathrm{Hb}$.

Enzymes and the active site. Almost all enzymes are proteins, and there are some nucleic acids that behave like enzymes - these are called ribozymes. An enzyme, like any protein, has a primary structure, the amino acid sequence, and also a secondary and a tertiary structure. When you look at a tertiary structure the chain criss-crosses itself and hence many crevices or pockets are made, and one such pocket is the active site. An active site of an enzyme is a crevice or pocket into which the substrate fits, and thus enzymes, through their active site, catalyse reactions at a high rate. Inorganic catalysts work efficiently at high temperatures and high pressures, while enzymes get damaged at high temperatures, say above $40^\circ\mathrm{C}$. Enzymes isolated from organisms that normally live under extremely high temperatures, for example hot vents and sulphur springs, are stable and retain their catalytic power even at high temperatures, up to $80^\circ$ to $90^\circ\mathrm{C}$, so thermal stability is an important quality of such enzymes isolated from thermophilic organisms.

Physical change, chemical reaction and rate. A physical change simply refers to a change in shape without breaking of bonds, and a change in state of matter, when ice melts into water or water becomes a vapour, is also a physical process. When bonds are broken and new bonds are formed during transformation, this will be called a chemical reaction - for example $$\mathrm{Ba(OH)_2 + H_2SO_4 \rightarrow BaSO_4 + 2H_2O}$$ an inorganic reaction, while hydrolysis of starch into glucose is an organic chemical reaction. Rate of a physical or chemical process refers to the amount of product formed per unit time, written as $\text{rate} = \frac{\delta P}{\delta t}$, and rate can also be called velocity if the direction is specified. A general rule of thumb is that rate doubles or decreases by half for every $10^\circ\mathrm{C}$ change in either direction.

How much faster. Catalysed reactions proceed at rates vastly higher than that of uncatalysed ones. In $$\mathrm{CO_2 + H_2O} \xrightarrow{\text{carbonic anhydrase}} \mathrm{H_2CO_3}$$ about 200 molecules of $\mathrm{H_2CO_3}$ are formed in an hour without any enzyme, but with carbonic anhydrase, the enzyme present within the cytoplasm, about 600,000 molecules are formed every second - the enzyme has accelerated the reaction rate by about 10 million times. There are thousands of types of enzymes, each catalysing a unique chemical or metabolic reaction. A multistep chemical reaction, when each of the steps is catalysed by the same enzyme complex or by different enzymes, is called a metabolic pathway, and $$\mathrm{C_6H_{12}O_6 + O_2 \rightarrow 2C_3H_4O_3 + 2H_2O}$$ is a metabolic pathway in which glucose becomes pyruvic acid through ten different enzyme catalysed metabolic reactions. The same pathway with one or two additional reactions gives lactic acid in our skeletal muscle under anaerobic conditions, pyruvic acid under normal aerobic conditions, and ethanol in yeast during fermentation.

Activation energy and the ES complex. The chemical which is converted into a product is called a substrate, and an enzyme converts a substrate $S$ into a product $P$. The substrate has to bind the enzyme at its active site within a given cleft or pocket, and has to diffuse towards the active site; there is thus an obligatory formation of an $ES$ complex, and this complex formation is a transient phenomenon. During the state where the substrate is bound to the enzyme active site, a new structure of the substrate called the transition state structure is formed, and very soon, after the expected bond breaking and making is completed, the product is released from the active site. All other intermediate structural states are unstable, and stability is related to the energy status of the molecule or structure. On the graph the y-axis represents the potential energy content and the x-axis the progression of the structural transformation through the transition state. If $P$ is at a lower level than $S$ the reaction is an exothermic reaction and one need not supply energy, by heating, in order to form the product. Whether it is an exothermic or spontaneous reaction, or an endothermic or energy requiring reaction, the $S$ has to go through a much higher energy state, or transition state, and the difference in average energy content of $S$ from that of this transition state is called the activation energy. Enzymes eventually bring down this energy barrier, making the transition of $S$ to $P$ more easy.

The catalytic cycle. Each enzyme $E$ has a substrate $S$ binding site in its molecule, so that a highly reactive enzyme-substrate complex $ES$ is produced, and this complex is short-lived and dissociates into its products $P$ and the unchanged enzyme, with an intermediate formation of the enzyme-product complex $EP$. The formation of the $ES$ complex is essential for catalysis. $$E + S \rightleftharpoons ES \rightarrow EP \rightarrow E + P$$ The four steps: (1) first, the substrate binds to the active site of the enzyme, fitting into the active site; (2) the binding of the substrate induces the enzyme to alter its shape, fitting more tightly around the substrate; (3) the active site of the enzyme, now in close proximity of the substrate, breaks the chemical bonds of the substrate, and the new enzyme-product complex is formed; (4) the enzyme releases the products of the reaction, and the free enzyme is ready to bind to another molecule of the substrate and run through the catalytic cycle once again.

Factors affecting enzyme activity. The activity of an enzyme can be affected by a change in the conditions which can alter the tertiary structure of the protein - temperature, pH, change in substrate concentration, and binding of specific chemicals that regulate its activity. Enzymes generally function in a narrow range of temperature and pH, and each enzyme shows its highest activity at a particular temperature and pH called the optimum temperature and optimum pH, activity declining both below and above the optimum value. Low temperature preserves the enzyme in a temporarily inactive state, whereas high temperature destroys enzymatic activity because proteins are denatured by heat. With the increase in substrate concentration the velocity of the enzymatic reaction rises at first, and the reaction ultimately reaches a maximum velocity, $V_{max}$, which is not exceeded by any further rise in the concentration of the substrate. This is because the enzyme molecules are fewer than the substrate molecules, and after saturation of these molecules there are no free enzyme molecules to bind with the additional substrate molecules. On the graph, $K_m$ marks the substrate concentration at which the velocity is half of $V_{max}$.

Inhibition. The activity of an enzyme is also sensitive to the presence of specific chemicals that bind to the enzyme, and when the binding of the chemical shuts off enzyme activity the process is called inhibition and the chemical is called an inhibitor. When the inhibitor closely resembles the substrate in its molecular structure and inhibits the activity of the enzyme, it is known as a competitive inhibitor; due to its close structural similarity with the substrate the inhibitor competes with the substrate for the substrate-binding site of the enzyme, consequently the substrate cannot bind and as a result the enzyme action declines. The standard example is the inhibition of succinic dehydrogenase by malonate, which closely resembles the substrate succinate in structure, and such competitive inhibitors are often used in the control of bacterial pathogens.

The six classes. Enzymes are divided into 6 classes, each with 4 to 13 subclasses, and named accordingly by a four-digit number. Oxidoreductases or dehydrogenases catalyse oxidoreduction between two substrates $S$ and $S'$. Transferases catalyse a transfer of a group $G$, other than hydrogen, between a pair of substrates. Hydrolases catalyse hydrolysis of ester, ether, peptide, glycosidic, C-C, C-halide or P-N bonds. Lyases catalyse removal of groups from substrates by mechanisms other than hydrolysis, leaving double bonds. Isomerases catalyse inter-conversion of optical, geometric or positional isomers. Ligases catalyse the linking together of 2 compounds, for example joining of C-O, C-S, C-N and P-O bonds.

Co-factors. Enzymes are composed of one or several polypeptide chains, however there are a number of cases in which non-protein constituents called co-factors are bound to the enzyme to make the enzyme catalytically active, and in these instances the protein portion of the enzyme is called the apoenzyme. Three kinds of cofactors may be identified - prosthetic groups, co-enzymes and metal ions. Prosthetic groups are organic compounds and are distinguished from other cofactors in that they are tightly bound to the apoenzyme - for example in peroxidase and catalase, which catalyse the breakdown of hydrogen peroxide to water and oxygen, haem is the prosthetic group and it is a part of the active site of the enzyme. Co-enzymes are also organic compounds but their association with the apoenzyme is only transient, usually occurring during the course of catalysis, and the essential chemical components of many coenzymes are vitamins - for example the coenzyme nicotinamide adenine dinucleotide, NAD, and NADP, which contain the vitamin niacin. A number of enzymes require metal ions for their activity, which form coordination bonds with side chains at the active site and at the same time form one or more coordination bonds with the substrate - for example zinc is a cofactor for the proteolytic enzyme carboxypeptidase. Catalytic activity is lost when the co-factor is removed from the enzyme, which testifies that they play a crucial role in the catalytic activity of the enzyme.

Tier 1 - Concept Checks

Question 1

Q. A living tissue is ground in trichloroacetic acid and the slurry is strained through cheesecloth. Name the two fractions you get and say what each holds.

Answer. The filtrate is the acid-soluble pool, and it holds thousands of organic compounds - the small ones. The retentate is the acid-insoluble fraction, and it holds proteins, nucleic acids, polysaccharides and lipids. The acid used is trichloroacetic acid, $\mathrm{Cl_3CCOOH}$.

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Question 2

Q. What is a biomolecule?

Answer. All the carbon compounds that we get from living tissues can be called biomolecules. The definition is that wide on purpose - it covers the small compounds of the acid-soluble pool and the macromolecules of the acid-insoluble fraction alike.

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Question 3

Q. Define wet weight, dry weight and ash.

Answer. Wet weight is the weight of a small amount of fresh living tissue, before anything is done to it. Dry weight is what remains after the tissue is dried and all the water evaporates. Ash is what is left after the tissue is fully burnt, when all the carbon compounds are oxidised to gaseous form - $\mathrm{CO_2}$ and water vapour - and are removed. The ash contains inorganic elements like calcium and magnesium.

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Question 4

Q. Which is the most abundant element in the human body, and which is the most abundant chemical in living organisms?

Answer. Oxygen is the most abundant element in the human body, at 65.0 per cent by weight. Water is the most abundant chemical in living organisms, at 70 to 90 per cent of the total cellular mass. Keep the two words apart - element and chemical compound are being asked in the same breath and the answers are different.

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Question 5

Q. Define an amino acid, and name the four groups attached to its alpha-carbon.

Answer. Amino acids are organic compounds containing an amino group and an acidic group as substituents on the same carbon, the $\alpha$-carbon - hence they are called $\alpha$-amino acids. They are substituted methanes, and the four substituent groups occupying the four valency positions are hydrogen, the carboxyl group $\mathrm{-COOH}$, the amino group $\mathrm{-NH_2}$, and a variable group designated as the R group.

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Question 6

Q. How many types of amino acids occur in proteins, and which amino acid does each of the R groups hydrogen, methyl and hydroxy methyl give?

Answer. Based on the nature of the R group there are many amino acids, but those which occur in proteins are only of twenty types. R group hydrogen gives glycine. A methyl group gives alanine. Hydroxy methyl gives serine.

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Question 7

Q. Give one acidic, one basic and one neutral amino acid, and name the three aromatic amino acids.

Answer. Acidic - glutamic acid. Basic - lysine. Neutral - valine. They are classified this way by the number of amino and carboxyl groups they carry. The aromatic amino acids are tyrosine, phenylalanine and tryptophan.

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Question 8

Q. What is the zwitterionic form of an amino acid?

Answer. At an intermediate pH the $\mathrm{-COOH}$ group loses a proton and the $\mathrm{-NH_2}$ group gains one at the same time, so the molecule carries both a positive and a negative charge on the same molecule while being electrically neutral overall. This is the zwitterionic form. It exists because the $\mathrm{-NH_2}$ and $\mathrm{-COOH}$ groups are ionizable, so the structure of amino acids changes in solutions of different pH.

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Question 9

Q. How many carbons are there in palmitic acid and in arachidonic acid, and what makes a fatty acid unsaturated?

Answer. Palmitic acid has 16 carbons including the carboxyl carbon. Arachidonic acid has 20 carbon atoms including the carboxyl carbon. A saturated fatty acid is without a double bond; an unsaturated fatty acid has one or more $\mathrm{C=C}$ double bonds.

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Question 10

Q. What is glycerol chemically, and what are monoglycerides, diglycerides and triglycerides?

Answer. Glycerol is trihydroxy propane. Many lipids have both glycerol and fatty acids, where the fatty acids are found esterified with glycerol. One fatty acid esterified gives a monoglyceride, two give a diglyceride and three give a triglyceride. These are also called fats and oils based on melting point - oils have a lower melting point, for example gingelly oil, and hence remain as oil in winters.

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Question 11

Q. What is a phospholipid, where is it found, and give one example.

Answer. Some lipids have phosphorous and a phosphorylated organic compound in them - these are phospholipids. They are found in the cell membrane. Lecithin is one example. Stated fully, phospholipids contain, in addition to glycerol and fatty acids, a phosphorylated nitrogenous compound. Some tissues, especially the neural tissues, have lipids with more complex structures.

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Question 12

Q. Name the five nitrogen bases and sort them into purines and pyrimidines.

Answer. The five are adenine, guanine, cytosine, uracil and thymine. Adenine and guanine are substituted purines. Cytosine, uracil and thymine are substituted pyrimidines. The skeletal heterocyclic rings themselves are called purine and pyrimidine respectively.

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Question 13

Q. What is a nucleoside and what is a nucleotide? Give the nucleoside and the nucleotide of adenine.

Answer. When a nitrogen base is found attached to a sugar, it is called a nucleoside. If a phosphate group is also found esterified to the sugar, it is called a nucleotide. For adenine, the nucleoside is adenosine and the nucleotide is adenylic acid. Nucleic acids like DNA and RNA consist of nucleotides only.

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Question 14

Q. What are primary metabolites? Name the categories that belong to them.

Answer. In animal tissues one notices the presence of all such categories of compounds, and these are primary metabolites. The categories are amino acids, sugars, fatty acids, glycerol, nucleotides, nucleosides and nitrogen bases. Primary metabolites have identifiable functions and play known roles in normal physiological processes.

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Question 15

Q. What are secondary metabolites, and do we know what all of them do?

Answer. When one analyses plant, fungal and microbial cells, one would see thousands of compounds other than the primary metabolites - alkaloids, flavonoids, rubber, essential oils, antibiotics, coloured pigments, scents, gums and spices - and these are called secondary metabolites. No - we do not at the moment understand the role or functions of all the secondary metabolites in host organisms. However, many of them are useful to human welfare - rubber, drugs, spices, scents and pigments - and some have ecological importance.

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Question 16

Q. Give the three molecular weight figures that divide micromolecules from macromolecules.

Answer. All the compounds found in the acid soluble pool have molecular weights ranging from 18 to around 800 daltons (Da) approximately. Those with molecular weights less than one thousand dalton are usually referred to as micromolecules or simply biomolecules. The classes of the acid insoluble fraction, with the exception of lipids, have molecular weights in the range of ten thousand daltons and above, and these are the macromolecules or biomacromolecules.

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Question 17

Q. Which four organic compounds make up the acid insoluble fraction, and which three of them comprise the true macromolecular fraction?

Answer. The acid insoluble fraction has only four types of organic compounds - proteins, nucleic acids, polysaccharides and lipids. But polysaccharides, polypeptides and polynucleotides together comprise the true macromolecular fraction of any living tissue or cell. Lipids travel with them but are not counted in that trio.

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Question 18

Q. Name the most abundant protein in the animal world and the most abundant protein in the whole of the biosphere.

Answer. Collagen is the most abundant protein in the animal world. Ribulose bisphosphate Carboxylase-Oxygenase (RuBisCO) is the most abundant protein in the whole of the biosphere. Collagen also serves as the intercellular ground substance.

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Question 19

Q. Are all enzymes proteins? What is the active site of an enzyme?

Answer. Almost all enzymes are proteins, and that is the exact wording. There are some nucleic acids that behave like enzymes - these are called ribozymes. When you look at a tertiary structure, the chain criss-crosses itself, and hence many crevices or pockets are made; one such pocket is the active site. An active site of an enzyme is a crevice or pocket into which the substrate fits, and thus enzymes, through their active site, catalyse reactions at a high rate.

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Question 20

Q. What is a co-factor, what is the apoenzyme, and what are the three kinds of co-factor?

Answer. Non-protein constituents called co-factors are bound to the enzyme to make the enzyme catalytically active, and the protein portion of the enzyme is then called the apoenzyme. Three kinds of cofactors may be identified - prosthetic groups, co-enzymes and metal ions. Prosthetic groups are organic compounds tightly bound to the apoenzyme, for example haem in peroxidase and catalase. Co-enzymes are organic compounds whose association with the apoenzyme is only transient, for example NAD and NADP, which contain the vitamin niacin. Metal ions form coordination bonds with side chains at the active site and with the substrate, for example zinc in carboxypeptidase. Catalytic activity is lost when the co-factor is removed from the enzyme.

Tier 2 - Application and Identification

Question 21

Q. A compound has an amino group and a carboxyl group attached to the same carbon, and its R group is a methyl group. Name it and give its class.

Answer. It is alanine. The class is an $\alpha$-amino acid, because amino acids are organic compounds containing an amino group and an acidic group as substituents on the same carbon, the $\alpha$-carbon. The R group is what names it - a methyl group as the R group gives alanine, while hydrogen would give glycine and hydroxy methyl would give serine.

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Question 22

Q. At a particular pH a molecule of glycine is found to carry a positive charge and a negative charge at the same time, yet the solution shows it to be electrically neutral. Name this form and explain how it arises.

Answer. It is the zwitterionic form. It arises because the $\mathrm{-NH_2}$ and $\mathrm{-COOH}$ groups of an amino acid are ionizable, so the structure of amino acids changes in solutions of different pH. At this intermediate pH the $\mathrm{-COOH}$ loses a proton, giving a negative charge, while the $\mathrm{-NH_2}$ gains a proton, giving a positive charge, so both charges sit on the same molecule and cancel out. Note that zwitterionic does not mean uncharged - it means both charges are present at once.

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Question 23

Q. A fatty acid isolated from a tissue has 20 carbon atoms counting the carboxyl carbon, and its chain carries several $\mathrm{C=C}$ double bonds. Name the acid and say whether it is saturated or unsaturated.

Answer. It is arachidonic acid, which has 20 carbon atoms including the carboxyl carbon. It is unsaturated, because unsaturated fatty acids have one or more $\mathrm{C=C}$ double bonds while saturated fatty acids are without a double bond. The other named example to keep beside it is palmitic acid, which has 16 carbons, again including the carboxyl carbon.

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Question 24

Q. Gingelly oil stays liquid through the winter while butter sets hard in the same room. Give the reason in one line, and say what both of them are chemically.

Answer. Oils have a lower melting point - for example gingelly oil, and hence it remains as oil in winters - while fats have the higher melting point and stay solid at the same temperature. Chemically both are the same kind of compound: fats and oils are glycerides in which fatty acids are esterified to glycerol. They differ only in melting point, not in the elements they contain and not in the number of hydroxyl groups on the glycerol.

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Question 25

Q. A compound is made of uracil joined to a sugar, with a phosphate group esterified to that sugar. Name the compound, and say whether it is a nucleoside or a nucleotide.

Answer. It is uridylic acid, and it is a nucleotide. When a nitrogen base is found attached to a sugar, it is called a nucleoside - that would be uridine here. If a phosphate group is also found esterified to the sugar, it is called a nucleotide, so the phosphate is what makes it uridylic acid. Note where the phosphate sits - it is esterified to the sugar, not to the nitrogen base.

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Question 26

Q. Lipids have molecular weights that do not exceed 800 Da, yet they are found in the acid insoluble fraction along with the macromolecules. Explain.

Answer. The reason is handling, not molecular weight. Lipids are indeed small molecular weight compounds, but they are present not only as such but also arranged into structures like the cell membrane and other membranes. When we grind a tissue we are disrupting the cell structure, so the cell membrane and other membranes are broken into pieces and form vesicles which are not water soluble. Therefore these membrane fragments, in the form of vesicles, get separated along with the acid insoluble pool. Lipids are thus not strictly macromolecules - they only travel with them.

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Question 27

Q. Two white powders are tested with iodine. One turns blue and the other shows no colour change. Identify both and explain the difference.

Answer. The one that turns blue is starch; the one that does not is cellulose. Starch forms helical secondary structures and can hold $\mathrm{I_2}$ molecules in the helical portion, and the starch-$\mathrm{I_2}$ complex is blue in colour. Cellulose does not contain complex helices and hence cannot hold $\mathrm{I_2}$. Say it carefully in the answer - starch does not react with iodine, it holds the $\mathrm{I_2}$ molecules in its helix.

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Question 28

Q. The hard outer covering of a prawn is analysed and found to be a polysaccharide built of modified sugars. Name it, and say whether such complex polysaccharides are homopolymers or heteropolymers.

Answer. It is chitin - exoskeletons of arthropods have a complex polysaccharide called chitin. These complex polysaccharides are mostly homopolymers, even though they have as building blocks amino-sugars and chemically modified sugars such as glucosamine and N-acetyl galactosamine. The modified building block does not make the polymer a heteropolymer, because the same modified unit repeats.

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Question 29

Q. A protein is found to be an assembly of four polypeptide subunits, two of one kind and two of another. Name the protein and the level of structure being described.

Answer. The protein is adult human haemoglobin, $\mathrm{Hb}$, and the level is the quaternary structure. Adult human haemoglobin consists of 4 subunits, two of which are identical to each other - two subunits of $\alpha$ type and two subunits of $\beta$ type. Quaternary structure is the manner in which these individual folded polypeptides or subunits are arranged with respect to each other, which is the architecture of a protein. It exists only in proteins that are an assembly of more than one polypeptide.

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Question 30

Q. An enzyme molecule releases its product and moments later is found bound to a fresh substrate molecule. Describe the cycle it has just been through, and say why it can repeat it.

Answer. It has run through the catalytic cycle, which has four steps.

1. First, the substrate binds to the active site of the enzyme, fitting into the active site.
2. The binding of the substrate induces the enzyme to alter its shape, fitting more tightly around the substrate.
3. The active site of the enzyme, now in close proximity of the substrate, breaks the chemical bonds of the substrate, and the new enzyme-product complex is formed.
4. The enzyme releases the products of the reaction, and the free enzyme is ready to bind to another molecule of the substrate and run through the catalytic cycle once again.

Written as an equation, $$E + S \rightleftharpoons ES \rightarrow EP \rightarrow E + P$$ It can repeat the cycle because the $ES$ complex dissociates into its products $P$ and the unchanged enzyme - the enzyme comes out of the cycle exactly as it went in. The formation of the $ES$ complex is essential for catalysis, and it is a transient phenomenon.

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Question 31

Q. One tube of an enzyme is kept in melting ice, a second is boiled, and a third holds an enzyme taken from a bacterium living in a sulphur spring and is held at $85^\circ\mathrm{C}$. Say what has happened to the enzyme in each tube.

Answer. In melting ice the enzyme is not destroyed - low temperature preserves the enzyme in a temporarily inactive state, and it works again when warmed. In the boiled tube the activity is gone for good - high temperature destroys enzymatic activity, because proteins are denatured by heat, and enzymes get damaged at high temperatures, say above $40^\circ\mathrm{C}$. The third tube still works. Enzymes isolated from organisms that normally live under extremely high temperatures, for example hot vents and sulphur springs, are stable and retain their catalytic power even at high temperatures, up to $80^\circ$ to $90^\circ\mathrm{C}$ - thermal stability is an important quality of such enzymes isolated from thermophilic organisms.

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Question 32

Q. In an experiment the substrate concentration is raised step by step. The velocity climbs, then flattens and will not rise further. Name the value it has reached and explain why more substrate does not help.

Answer. It has reached $V_{max}$, the maximum velocity, which is not exceeded by any further rise in the concentration of the substrate. With the increase in substrate concentration the velocity of the enzymatic reaction rises at first, and then it stops rising. The reason is not that the substrate runs out - it is because the enzyme molecules are fewer than the substrate molecules, and after saturation of these molecules there are no free enzyme molecules to bind with the additional substrate molecules. On the same graph, the substrate concentration at which the velocity is half of $V_{max}$ is marked $K_m$.

Tier 3 - Comparisons and Long Answers

Question 33

Q. Distinguish between a micromolecule and a macromolecule.

Answer.

Feature	Micromolecule	Macromolecule
Other name	Simply biomolecule	Biomacromolecule
Molecular weight	Less than one thousand dalton; the pool runs from 18 to around 800 Da	Ten thousand daltons and above
Where it is found	The acid-soluble pool, the filtrate	The acid-insoluble fraction, the retentate
Nature	Not polymeric	Polymeric substances, with the exception of lipids
Examples	Amino acids, sugars, fatty acids, glycerol, nucleotides, nucleosides, nitrogen bases	Proteins, nucleic acids, polysaccharides - and lipids, which travel with them
What it represents	Roughly the cytoplasmic composition	Macromolecules from cytoplasm and organelles

Together the two represent the entire chemical composition of living tissues or organisms. The one line that decides the question: biomolecules with molecular weights less than one thousand dalton are micromolecules, and those found in the acid insoluble fraction are macromolecules.

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Question 34

Q. Distinguish between primary and secondary metabolites.

Answer.

Feature	Primary metabolites	Secondary metabolites
Where they are noticed	In animal tissues	In plant, fungal and microbial cells
Function	Have identifiable functions and play known roles in normal physiological processes	We do not at the moment understand the role or functions of all of them in host organisms
Examples	Amino acids, sugars, fatty acids, glycerol, nucleotides, nucleosides, nitrogen bases	Alkaloids, flavonoids, rubber, essential oils, antibiotics, coloured pigments, scents, gums, spices
Use to humans	Used by the body itself in its normal working	Many are useful to human welfare - rubber, drugs, spices, scents and pigments; some have ecological importance

Classes of secondary metabolites with examples: pigments - carotenoids, anthocyanins; alkaloids - morphine, codeine; terpenoides - monoterpenes, diterpenes; essential oils - lemon grass oil; toxins - abrin, ricin; lectins - concanavalin A; drugs - vinblastin, curcumin; polymeric substances - rubber, gums, cellulose.

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Question 35

Q. Distinguish between a homopolymer and a heteropolymer, and say which one a protein is and why.

Answer.

Feature	Homopolymer	Heteropolymer
Monomers	Only one type of monomer, repeating "n" number of times	More than one type of monomer in the same chain
Standard example	Cellulose - a polymeric polysaccharide consisting of only one type of monosaccharide, glucose	A protein - a polymer of amino acids

A protein is a heteropolymer and not a homopolymer, because there are 20 types of amino acids in it - alanine, cysteine, proline, tryptophan, lysine and the rest. Proteins are polypeptides, that is linear chains of amino acids linked by peptide bonds. Note also that the complex polysaccharides such as chitin are mostly homopolymers, even though their building blocks are amino-sugars and chemically modified sugars.

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Question 36

Q. Compare starch, cellulose and glycogen.

Answer.

Feature	Starch	Cellulose	Glycogen
Where it occurs	Plant tissues	Plant cell walls; also paper made from plant pulp and cotton fibre	Animals
Role	A store house of energy in plant tissues	Structural - plant cell walls are made of cellulose	The energy store animals have - the variant animals have
Building block	Glucose	Only one type of monosaccharide, glucose - so a homopolymer	Glucose
Secondary structure	Forms helical secondary structures	Does not contain complex helices	Branched, as the cartoon of glycogen shows
Iodine test	Can hold $\mathrm{I_2}$ molecules in the helical portion; the starch-$\mathrm{I_2}$ complex is blue in colour	Cannot hold $\mathrm{I_2}$, so no blue colour	Not the standard test material

Two extra points that get asked with this group. In a polysaccharide chain, say glycogen, the right end is called the reducing end and the left end is called the non-reducing end. And keep inulin apart from all three - inulin is a polymer of fructose, not of glucose.

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Question 37

Q. Distinguish between a purine and a pyrimidine.

Answer.

Feature	Purine	Pyrimidine
What the word names	The skeletal heterocyclic ring	The skeletal heterocyclic ring
Bases built on it	Adenine and guanine are substituted purines	Cytosine, uracil and thymine are substituted pyrimidines
Number of bases	Two	Three

Living organisms have a number of carbon compounds in which heterocyclic rings can be found, and the nitrogen bases are among them. The word substituted carries a mark of its own - the bare ring is the purine or the pyrimidine, and the base is that ring with groups substituted on to it. Learn the two purines as a pair, and everything left over is a pyrimidine.

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Question 38

Q. Distinguish between DNA and RNA.

Answer.

Feature	DNA	RNA
Full name	Deoxyribonucleic acid	Ribonucleic acid
Sugar	2-deoxyribose	Ribose, a monosaccharide pentose
What decides the name	A nucleic acid containing deoxyribose is called DNA	One which contains ribose is called RNA
Nitrogen bases	Adenine, guanine, cytosine and thymine	Adenine, guanine, cytosine and uracil
Building block	Nucleotides only	Nucleotides only
Role	Functions as genetic material	Functions as genetic material

Both are polynucleotides, and a nucleotide has three chemically distinct components - a heterocyclic compound, a monosaccharide, and a phosphoric acid or phosphate. Nucleic acids serve as genetic material; they carry hereditary information and are passed on from parental generation to progeny. The definition of DNA and RNA is built on the sugar and nothing else.

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Question 39

Q. Describe the four levels of protein structure.

Answer. Biologists describe the protein structure at four levels - primary, secondary, tertiary and quaternary.

Level	What it is
Primary	The sequence of amino acids, that is the positional information in a protein - which is the first amino acid, which is second, and so on
Secondary	The thread is folded in the form of a helix, similar to a revolving staircase, with other regions folded into other forms
Tertiary	The long protein chain is also folded upon itself like a hollow woolen ball
Quaternary	The manner in which individual folded polypeptides or subunits are arranged with respect to each other

Primary structure. A protein is imagined as a line, the left end represented by the first amino acid and the right end by the last. The first amino acid is also called the N-terminal amino acid and the last is called the C-terminal amino acid. There is no folding at this level at all - only the order in which the amino acids come.

Secondary structure. A protein thread does not exist throughout as an extended rigid rod. Of course, only some portions of the protein thread are arranged in the form of a helix, and in proteins, only right handed helices are observed. The two named forms are the $\alpha$-helix and the $\beta$-pleated sheet.

Tertiary structure. This gives us a three dimensional view of a protein, and it is absolutely necessary for the many biological activities of proteins. It is the level at which the criss-crossing chain makes the crevices and pockets, one of which is the active site of an enzyme.

Quaternary structure. Some proteins are an assembly of more than one polypeptide or subunits, and their arrangement - a linear string of spheres, or spheres arranged one upon each other in the form of a cube or plate - is the architecture of a protein. Adult human haemoglobin consists of 4 subunits, two of $\alpha$ type and two of $\beta$ type.

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Question 40

Q. Distinguish between a nucleoside and a nucleotide, with examples of each.

Answer.

Feature	Nucleoside	Nucleotide
Composition	A nitrogen base attached to a sugar	A nitrogen base attached to a sugar, with a phosphate group esterified to the sugar
Phosphate	Absent	Present, esterified to the sugar
Examples	Adenosine, guanosine, thymidine, uridine, cytidine	Adenylic acid, thymidylic acid, guanylic acid, uridylic acid, cytidylic acid
In nucleic acids	Not the unit of the chain	Nucleic acids like DNA and RNA consist of nucleotides only

Nitrogen base	Nucleoside	Nucleotide
Adenine	Adenosine	Adenylic acid
Guanine	Guanosine	Guanylic acid
Cytosine	Cytidine	Cytidylic acid
Uracil	Uridine	Uridylic acid
Thymine	Thymidine	Thymidylic acid

The build-up runs nitrogen base, plus sugar gives a nucleoside, plus phosphate esterified to the sugar gives a nucleotide. Read the ending of the name in the options - the ones ending in "-osine" or "-idine" are nucleosides, and the ones that are an "acid" are nucleotides.

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Question 41

Q. How does an enzyme differ from an inorganic catalyst?

Answer.

Feature	Inorganic catalyst	Enzyme
Chemical nature	An inorganic substance	Almost all enzymes are proteins; some nucleic acids behave like enzymes and are called ribozymes
Working conditions	Work efficiently at high temperatures and high pressures	Get damaged at high temperatures, say above $40^\circ\mathrm{C}$
The exception	Not applicable	Enzymes from organisms of hot vents and sulphur springs are stable up to $80^\circ$ to $90^\circ\mathrm{C}$ - thermal stability
Structure that does the work	No active site of this kind	A crevice or pocket made by the criss-crossing tertiary structure - the active site into which the substrate fits
Specificity	Generally acts on many reactions	There are thousands of types of enzymes, each catalysing a unique chemical or metabolic reaction
pH range	Wide	Enzymes generally function in a narrow range of temperature and pH, with an optimum temperature and optimum pH

What both of them do. Catalysed reactions proceed at rates vastly higher than that of uncatalysed ones, and enzymes eventually bring down the energy barrier - the activation energy - making the transition of $S$ to $P$ more easy. Activation energy is the difference in average energy content of $S$ from that of the transition state, so the enzyme lowers the hump without moving $S$ or $P$.

The number that shows the scale of it. In $$\mathrm{CO_2 + H_2O} \xrightarrow{\text{carbonic anhydrase}} \mathrm{H_2CO_3}$$ in the absence of any enzyme about 200 molecules of $\mathrm{H_2CO_3}$ are formed in an hour, while with carbonic anhydrase about 600,000 molecules are formed every second - an acceleration of about 10 million times.

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Question 42

Q. Explain competitive inhibition with an example.

Answer. The activity of an enzyme is sensitive to the presence of specific chemicals that bind to the enzyme. When the binding of the chemical shuts off enzyme activity, the process is called inhibition and the chemical is called an inhibitor.

When the inhibitor closely resembles the substrate in its molecular structure and inhibits the activity of the enzyme, it is known as a competitive inhibitor. The mechanism follows from that resemblance in three steps.

1. Due to its close structural similarity with the substrate, the inhibitor competes with the substrate for the substrate-binding site of the enzyme.
2. Consequently the substrate cannot bind.
3. As a result the enzyme action declines.

The standard example.

Enzyme inhibited	Succinic dehydrogenase
The real substrate	Succinate
The competitive inhibitor	Malonate, which closely resembles succinate in structure

Such competitive inhibitors are often used in the control of bacterial pathogens. Note that the inhibitor works by occupying the active site, which is a crevice or pocket into which the substrate fits - it is the resemblance, not any chemical attack, that does the blocking.

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Question 43

Q. Enzymes are divided into six classes. Name them and give the reaction each one catalyses.

Answer. Thousands of enzymes have been discovered, isolated and studied, and most of these enzymes have been classified into different groups based on the type of reactions they catalyse. Enzymes are divided into 6 classes, each with 4 to 13 subclasses, and named accordingly by a four-digit number.

Class	The reaction it catalyses
1. Oxidoreductases, also called dehydrogenases	Oxidoreduction between two substrates $S$ and $S'$
2. Transferases	Transfer of a group $G$, other than hydrogen, between a pair of substrates $S$ and $S'$
3. Hydrolases	Hydrolysis of ester, ether, peptide, glycosidic, C-C, C-halide or P-N bonds
4. Lyases	Removal of groups from substrates by mechanisms other than hydrolysis, leaving double bonds
5. Isomerases	Inter-conversion of optical, geometric or positional isomers
6. Ligases	Linking together of 2 compounds, for example joining of C-O, C-S, C-N and P-O bonds

The example reaction for the first two classes, written out.

Oxidoreductases: $$S_{\text{reduced}} + S'_{\text{oxidised}} \rightarrow S_{\text{oxidised}} + S'_{\text{reduced}}$$

Transferases: $$S - G + S' \rightarrow S + S' - G$$

The pair most often confused is hydrolases against lyases - hydrolases work by hydrolysis, lyases remove groups by mechanisms other than hydrolysis and leave double bonds behind. Remember that ligases link and isomerases inter-convert isomers.

The Chapter-End Exercises

All eleven exercises at the end of this chapter are answered somewhere in this chapter. The table shows where each one is worked out, so you can check your own attempt against a full answer. Eight of them are answered inside the teaching sections, and the three activity-type exercises are worked out below.

Exercise	Answered as
1. What are macromolecules? Give examples	Question 4 of the Biomacromolecules section
2. What is meant by tertiary structure of proteins	Question 10 of the Structure of Proteins section
3. Structures of 10 interesting small molecular weight biomolecules, the industry and the buyers	Question 44 below, since it is not covered anywhere else
4. List of proteins used as therapeutic agents, and other applications of proteins	Question 13 of the Proteins section
5. Explain the composition of triglyceride	Question 7 of the Lipids section
6. Building models of biomolecules with ball and stick models	Question 45 below, since it is not covered anywhere else
7. Draw the structure of the amino acid, alanine	Question 6 of the Amino Acids section
8. What are gums made of? Is Fevicol different	Question 11 of the Primary and Secondary Metabolites section
9. A qualitative test for proteins, fats and oils and amino acids, tested on fruit juice, saliva, sweat and urine	Question 46 below, since it is not covered anywhere else
10. How much cellulose is made by all the plants in the biosphere, compared with paper	Question 13 of the Polysaccharides section
11. Describe the important properties of enzymes	Question 15 of the Factors Affecting Enzyme Activity, Classification and Co-factors section

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Question 44

Q. Find and write down structures of 10 interesting small molecular weight biomolecules. Find if there is any industry which manufactures the compounds by isolation. Find out who are the buyers. This is one of the chapter-end exercises.

Answer. Every compound below is a micromolecule - a biomolecule with a molecular weight of less than one thousand dalton, sitting in the acid-soluble pool, whose compounds run from 18 to around 800 Da. Ten of them, taken from this chapter itself, with the class and the structure written on one line.

Compound	Class	Formula or structure
Glucose	Monosaccharide sugar	$\mathrm{C_6H_{12}O_6}$ - a six carbon sugar, the monomer of cellulose, starch and glycogen
Ribose	Monosaccharide pentose	$\mathrm{C_5H_{10}O_5}$ - the five carbon sugar of RNA; 2-deoxyribose is the same ring with one oxygen less, and it gives DNA
Glycine	Amino acid, R group is hydrogen	$\mathrm{NH_2-CH_2-COOH}$, that is $\mathrm{C_2H_5NO_2}$
Alanine	Amino acid, R group is a methyl group	$\mathrm{CH_3-CH(NH_2)-COOH}$, that is $\mathrm{C_3H_7NO_2}$
Serine	Amino acid, R group is hydroxy methyl	$\mathrm{HO-CH_2-CH(NH_2)-COOH}$, that is $\mathrm{C_3H_7NO_3}$
Palmitic acid	Saturated fatty acid	$\mathrm{CH_3(CH_2)_{14}COOH}$, that is $\mathrm{C_{16}H_{32}O_2}$ - 16 carbons including the carboxyl carbon, no double bond
Arachidonic acid	Unsaturated fatty acid	$\mathrm{C_{20}H_{32}O_2}$ - 20 carbon atoms including the carboxyl carbon, with several $\mathrm{C=C}$ double bonds
Glycerol	Simple lipid, trihydroxy propane	$\mathrm{CH_2OH-CHOH-CH_2OH}$, that is $\mathrm{C_3H_8O_3}$ - three carbons carrying three hydroxyl groups
Adenine	Nitrogen base, a substituted purine	$\mathrm{C_5H_5N_5}$ - the purine heterocyclic ring with an amino group substituted on it
Uracil	Nitrogen base, a substituted pyrimidine	$\mathrm{C_4H_4N_2O_2}$ - the pyrimidine heterocyclic ring with two oxygen atoms substituted on it

Two more that are worth adding, because they show how the small ones are built up. Adenosine is the nucleoside - adenine attached to ribose. Adenylic acid is the nucleotide - adenine plus ribose plus a phosphate group esterified to the sugar, the phosphate coming from phosphoric acid, $\mathrm{H_3PO_4}$.

Is there an industry that manufactures them? Yes, and it works in three different ways.

How it is made	Compounds made that way
Isolation from a natural source	Palmitic acid, isolated from palm oil and other vegetable oils. Glycerol, recovered as a by-product when fats and oils are split during soap manufacture and biodiesel manufacture.
Hydrolysis of a natural polymer	Glucose, made on a very large scale by the hydrolysis of starch - the same reaction the chapter names as an organic chemical reaction, hydrolysis of starch into glucose.
Fermentation by microbes, or chemical synthesis	Amino acids such as glutamic acid and lysine, made by bacterial fermentation. Glycine, made by chemical synthesis. Nucleotides such as the guanylic and inosinic acid salts, made by fermentation and used as flavour enhancers.

Who buys them.

• The food industry - glucose as a sweetener and energy source, glutamic acid salts and nucleotides as flavour enhancers, glycerol as a humectant that keeps food moist.
• The pharmaceutical industry - amino acids for intravenous nutrition drips and for medicines, glycerol as a base for syrups and ointments.
• The cosmetic and soap industry - glycerol in creams and lotions, palmitic acid and its salts in soaps and cleansers.
• The animal feed industry - lysine and other amino acids added to poultry and cattle feed, because essential amino acids have to be supplied through the diet.
• Laboratories and research - purified amino acids, sugars, bases and nucleotides sold as laboratory reagents, and NAD and NADP as coenzyme reagents.

The general point behind the exercise is the one the chapter opens with: the most exciting aspect of chemistry deals with isolating thousands of compounds, small and big, from living organisms, determining their structure and if possible synthesising them. Industry does exactly that at scale.

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Question 45

Q. Can you attempt building models of biomolecules using commercially available atomic models (Ball and Stick models). This is one of the chapter-end exercises.

Answer. Yes, and the ball and stick set is well suited to the small biomolecules of this chapter, because the micromolecules have molecular weights of less than one thousand dalton and can be built with a handful of balls. Build them in this order, because each one teaches the next.

1. An amino acid - build this one first. Amino acids are substituted methanes. Start by building methane, $\mathrm{CH_4}$ - one carbon ball with four sticks going out to four hydrogens. Then pull off three of the hydrogens and put in their place the carboxyl group $\mathrm{-COOH}$, the amino group $\mathrm{-NH_2}$, and the R group, leaving one hydrogen where it was. What the model shows you straight away is that there are four substituent groups occupying the four valency positions of the $\alpha$-carbon, and that all four sit on the same carbon - which is the whole definition. Swap the R group and you have moved from one amino acid to another: hydrogen gives glycine, a methyl group gives alanine, hydroxy methyl gives serine.

2. Glycerol - trihydroxy propane. Build a three carbon chain and attach one hydroxyl group to each carbon. The model makes the number three obvious, and that is the number that matters - three hydroxyl positions, so one fatty acid esterified gives a monoglyceride, two give a diglyceride, and three give a triglyceride.

3. A saturated fatty acid beside an unsaturated one. Build a carboxyl group $\mathrm{-COOH}$ attached to a chain of $\mathrm{-CH_2}$ groups. Make one chain saturated - without a double bond - and it lies out as a straight, freely turning zig-zag. Now build a second chain and put in one $\mathrm{C=C}$ double bond using the double stick. The double bond will not turn, and it puts a visible kink in the chain. That kink, which you can see and feel in your hands, is the reason oils have a lower melting point - kinked chains cannot pack together as closely as straight ones - and hence gingelly oil remains as oil in winters.

4. A nucleotide. Build it in the three parts the chapter names. A heterocyclic compound - the nitrogenous base, a ring carrying nitrogen atoms in it. A monosaccharide - the sugar, ribose or 2-deoxyribose. A phosphoric acid or phosphate. Join the base to the sugar and you have a nucleoside; then esterify the phosphate to the sugar and you have a nucleotide. Building it in that order fixes the one thing students lose marks on - the phosphate goes on to the sugar, not on to the base.

What a ball and stick model shows well.

• Bond angles and the shape around each atom - especially the tetrahedral carbon, with its four valency positions pointing away from each other.
• The difference a double bond makes - a single bond can rotate, a $\mathrm{C=C}$ double bond cannot, so the chain acquires a fixed kink.
• Which group is attached to which atom - so the amino group and the carboxyl group on the same carbon, and the phosphate on the sugar, become things you can see rather than things you memorise.
• The count of atoms in a small molecule, so 16 carbons in palmitic acid and 20 in arachidonic acid, in both counting the carboxyl carbon, stop being abstract.

What it cannot show.

• The folding of a protein. A protein thread does not exist throughout as an extended rigid rod - it is folded in the form of a helix, only some portions of the thread are arranged that way, and the chain is further folded upon itself like a hollow woolen ball. No hand-built stick model can hold the secondary and tertiary structure of even a small protein, and tertiary structure is absolutely necessary for the many biological activities of proteins.
• The size of a macromolecule. Macromolecules are ten thousand daltons and above, so a protein or a polysaccharide would need thousands of balls, while the micromolecules you can actually build are 18 to around 800 Da.
• The quaternary architecture - the manner in which folded subunits are arranged with respect to each other, as in the four subunits of haemoglobin.
• Anything that moves - the transient $ES$ complex, or the way the binding of the substrate induces the enzyme to alter its shape.

So the honest summary is that ball and stick models are excellent for the micromolecules and useless for the macromolecules, which is a useful thing to have discovered for yourself.

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Question 46

Q. Find out a qualitative test for proteins, fats and oils, amino acids and test any fruit juice, saliva, sweat and urine for them. This is one of the chapter-end exercises.

Answer. The standard qualitative tests, with what each one actually detects.

Looking for	Test	Reagent and how it is done	Positive result
Proteins	The biuret test	Add dilute sodium hydroxide to the sample, then a few drops of dilute copper sulphate solution	A violet or purple colour. It detects the peptide bond, which is exactly what a protein is built of - proteins are polypeptides, linear chains of amino acids linked by peptide bonds
Fats and oils	The translucent spot test	Rub a drop of the sample on a piece of plain paper and let it dry	A translucent spot that does not dry off, because lipids are generally water insoluble and do not evaporate
Fats and oils	The Sudan III stain	Add a few drops of Sudan III to the sample and shake	The fat layer takes up a red colour, because the dye is fat soluble and collects in the lipid
Amino acids	The ninhydrin test	Add ninhydrin solution to the sample and warm it gently	A purple colour. It detects the free amino group of a free amino acid
Reducing sugars	Benedict's test	Add Benedict's solution and heat in a water bath	A brick red precipitate. Worth including here because fruit juice and urine are the obvious samples for it
Starch	The iodine test	Add a drop of iodine solution	A blue-black colour, because starch forms helical secondary structures and can hold $\mathrm{I_2}$ molecules in the helical portion, and the starch-$\mathrm{I_2}$ complex is blue in colour. Cellulose does not contain complex helices and hence cannot hold $\mathrm{I_2}$

What a student would actually expect from each sample.

Sample	Reducing sugar	Protein	Amino acid	Fat
Fruit juice	Strongly positive - fruit juice is rich in sugars	Usually negative or very faint	Faintly positive in some fruits	Negative
Saliva	Usually negative to start with, though it will turn positive if starch has been chewed, since saliva begins the hydrolysis of starch into glucose	Positive - saliva contains enzymes, and almost all enzymes are proteins	Faintly positive	Negative
Sweat	Negative	Negative or very faint	Positive - sweat carries free amino acids and urea	Negative
Urine	Normally negative	Normally negative	Faintly positive - small amounts of amino acids are excreted	Negative

How to read the results. The tests are qualitative - they tell you whether a class of biomolecule is present, not how much of it there is. A colour that appears means the group being tested for is there, and the group, not the whole compound, is what the reagent finds - the biuret test finds the peptide bond, the ninhydrin test finds the free amino group, and iodine finds the helix of starch.

One caution, and it matters. A positive protein or sugar result in a urine sample is a medical finding, not a conclusion to be drawn from a school laboratory test. Anyone getting such a result should have it checked by a doctor with a proper clinical test, because a school test can give a false positive from a dirty tube, a stale sample or a wrongly made reagent. The point of the exercise is to learn the tests and see the colours, not to diagnose anybody.