Biomolecules

The chapter begins with a question and a method. What is living tissue made of chemically? The method is simple enough to picture in the exam. Take a living tissue (a vegetable, a piece of liver, a microbial culture), grind it in trichloroacetic acid (Cl₃CCOOH) using a mortar and pestle, and strain the thick slurry through cheesecloth or cotton. You get two parts. The filtrate is the acid-soluble pool; the material left behind, the retentate, is the acid-insoluble fraction. In the acid-soluble pool are the small organic compounds, thousands of them, with molecular weights ranging from 18 to around 800 daltons (Da). These are the micromolecules or, loosely, the biomolecules: amino acids, sugars, fatty acids, glycerol, nitrogen bases, nucleosides, nucleotides. The acid-insoluble fraction contains only four types of organic compound: proteins, nucleic acids, polysaccharides and lipids, with molecular weights of ten thousand daltons and above (except lipids, which is a point examined separately in 9.3). The acid-soluble pool roughly represents the composition of the cytoplasm; the macromolecules come from the cytoplasm and the organelles together. Both together give the total organic content of the tissue.

To find the inorganic content, a different route is taken. Take a small weighed sample, dry it fully and weigh it; the loss is water. Then burn the dried sample completely so that every carbon compound is oxidised to gaseous CO₂ and water vapour and driven off. What remains is ash, which contains the inorganic elements (calcium, magnesium and so on) and the inorganic compounds (sulphate, phosphate and so on). Elemental analysis of the tissue shows carbon, hydrogen, oxygen, nitrogen, sulphur, phosphorus and so on; analysis of the organic compounds shows which compounds are present.

Two NCERT tables come straight into MCQs. Table 9.1, average composition of cells as a percentage of total cellular mass: water 70 to 90, proteins 10 to 15, carbohydrates 3, lipids 2, nucleic acids 5 to 7, ions 1. Water is the most abundant chemical in a living organism. Table 9.2 compares the earth's crust and the human body (per cent weight): hydrogen 0.14 versus 0.5; carbon 0.03 versus 18.5; oxygen 46.6 versus 65.0; nitrogen very little versus 3.3; sulphur 0.03 versus 0.3; sodium 2.8 versus 0.2; calcium 3.6 versus 1.5; magnesium 2.1 versus 0.1; silicon 27.7 versus negligible. Two conclusions the examiner wants: all the major elements of the crust are present in living things, but living organisms have far more carbon, hydrogen and oxygen per unit mass, because they are built of organic compounds; and silicon, abundant in the crust, is negligible in the body.

Amino acids are organic compounds containing an amino group (−NH₂) and an acidic carboxyl group (−COOH) as substituents on the same carbon, the α-carbon; hence they are called α-amino acids. NCERT describes them as substituted methanes: the α-carbon has four valency positions, occupied by a hydrogen atom, the carboxyl group, the amino group and a variable group called the R group. Amino acids differ only in R. In the proteinaceous amino acids R can be hydrogen (glycine), a methyl group −CH₃ (alanine), a hydroxymethyl group −CH₂OH (serine) and so on. There are twenty types of amino acids in proteins. The physical and chemical properties of an amino acid come from its three functional parts: the amino group, the carboxyl group and the R group.

Classification you must reproduce. Based on the number of amino and carboxyl groups there are acidic amino acids (an extra carboxyl group, e.g. glutamic acid), basic amino acids (an extra amino group, e.g. lysine) and neutral amino acids (one of each, e.g. valine). There are also aromatic amino acids with a ring in R: tyrosine, phenylalanine and tryptophan. Amino acids the body cannot make and must take in the diet are essential; the rest are non-essential (this distinction is used again in 9.4).

A particular property is the ionisable nature of −NH₂ and −COOH. In solution, depending on pH, the carboxyl group can lose a proton to become −COO⁻ and the amino group can gain one to become −NH₃⁺. So the structure of an amino acid changes with pH: at low pH it exists largely as the positively charged form, at high pH as the negatively charged form, and in between as a form carrying both charges at once, the zwitterionic form, in which the amino group is −NH₃⁺ and the carboxyl group is −COO⁻ on the same molecule. This is the answer to the exemplar MCQ and it also explains exercise 9.10, where titrating an amino acid against a weak base reveals the number of ionisable groups.

Lipids are generally water-insoluble. They could be simple fatty acids: a carboxyl group attached to an R group, where R could be a methyl, an ethyl or a higher number of −CH₂ groups, from 1 carbon up to 19 carbons. Two examples NCERT names with their carbon counts: palmitic acid has 16 carbons including the carboxyl carbon, and arachidonic acid has 20 carbon atoms including the carboxyl carbon. Fatty acids may be saturated (no double bonds in R) or unsaturated (one or more C=C double bonds). Another simple lipid is glycerol, which is trihydroxy propane, three carbons each carrying an −OH.

Many lipids have both fatty acids and glycerol. The fatty acids are esterified with glycerol (the −COOH of the fatty acid with an −OH of glycerol, an ester bond with loss of water) to give monoglycerides, diglycerides and triglycerides depending on whether one, two or three −OH groups are esterified. A triglyceride is glycerol joined to three fatty acids by three ester bonds; this is the whole answer to exercise 9.7. These are also called fats and oils, the difference being the melting point: oils have a lower melting point and are liquid at room temperature (NCERT's example is gingely oil), fats are solid. Oils are rich in unsaturated fatty acids, which is why they stay liquid.

Some lipids contain phosphorus and a phosphorylated organic compound; these are phospholipids, found in the cell membrane. NCERT's example is lecithin. Some tissues, especially neural tissue, have lipids with more complex structures. Keep two exam points ready: lipids are not strictly macromolecules (their molecular weight does not exceed 800 Da) but they appear in the acid-insoluble fraction, and the reason is in section 9.3; and lipids give the 'grease spot' qualitative test (exercise 9.13), while proteins give the Biuret test and amino acids the ninhydrin test.

Living organisms also contain heterocyclic carbon compounds, ring compounds in which one or more ring atoms is nitrogen. The ones that matter here are the nitrogen bases: adenine, guanine, cytosine, uracil and thymine. They fall into two families. The purines have a double ring (a six-membered ring fused to a five-membered ring): adenine and guanine. The pyrimidines have a single six-membered ring: cytosine, uracil and thymine. A memory aid students use is that the two shorter words (adenine, guanine) are the bigger molecules, and CUT (cytosine, uracil, thymine) are the pyrimidines.

Now the two definitions that are asked every single year. A nitrogen base attached to a sugar is a nucleoside. A nucleoside attached to a phosphate group is a nucleotide; so a nucleotide has three parts, base + sugar + phosphate. The sugar is a pentose, ribose in RNA nucleotides and 2'-deoxyribose in DNA nucleotides. The names you must be able to list: the nucleosides are adenosine, guanosine, thymidine, uridine and cytidine; the corresponding nucleotides are adenylic acid, guanylic acid, thymidylic acid, uridylic acid and cytidylic acid (in modern naming AMP, GMP and so on). DNA and RNA are polynucleotides, chains of nucleotides; the nucleotides are also the chemical currency of the cell in the form of ATP, NAD and so on.

The exam pattern is always the same: a 2-mark 'distinguish nucleoside from nucleotide with an example of each', a 1-mark MCQ 'nucleoside + phosphate = ?' and the exemplar long answer 'differentiate between nucleotide and nucleoside with two examples of each, along with structures'. For the structures, draw the pentose ring with carbons 1' to 5', put the base on C-1' and, for a nucleotide, the phosphate on C-5'.

The most exciting aspect of chemistry, NCERT says, is the enormous variety of organic compounds, and the organic compounds found in living tissue are the metabolites. In animal tissues you find amino acids, sugars, fatty acids, glycerol, nucleotides, nucleosides, nitrogen bases and so on, all of which have identifiable functions in physiological processes and are called primary metabolites. But when you analyse plant, fungal and microbial cells you also find thousands of other compounds: alkaloids, flavonoids, rubber, essential oils, antibiotics, coloured pigments, scents, gums, spices. These are secondary metabolites. Two features define them in the exam: they are found in plant, fungal and microbial cells (not typically in animal cells), and not all of them have a known role in the organism's physiology, though many are useful to human welfare (rubber, drugs, spices, scents, pigments). Some have ecological importance, for example as defences against herbivores or as attractants.

NCERT's Table 9.3 is the source of every question on this section, so learn one or two examples per row. Pigments: carotenoids, anthocyanins. Alkaloids: morphine, codeine. Terpenoids: monoterpenes, diterpenes. Essential oils: lemon grass oil. Toxins: abrin, ricin. Lectins: concanavalin A. Drugs: vinblastin, curcumin. Polymeric substances: rubber, gums, cellulose. Typical questions are 'classify the following as primary or secondary metabolites', 'name two secondary metabolites that are drugs' and the exemplar item 'classify penicillin, sulphonamide, vitamin C and growth hormone as natural or synthetic' (penicillin is a natural product from a fungus, vitamin C and growth hormone are natural, sulphonamide is synthetic).

Write the distinction in two clean lines: primary metabolites have identifiable functions in physiological processes and are present in all cells (amino acids, sugars, nucleotides); secondary metabolites are products of pathways not directly involved in growth, development or reproduction, mostly in plants, fungi and microbes, with functions not always known (alkaloids, terpenoids, pigments, toxins, lectins).

The acid-soluble pool has compounds with molecular weights below about one thousand daltons, the micromolecules. The acid-insoluble fraction has only four types of organic compound: proteins, nucleic acids, polysaccharides and lipids, and except for lipids these have molecular weights of ten thousand daltons and above. These large molecules are the biomacromolecules. A macromolecule is a polymer: a long chain formed by linking many small repeating units (monomers) by covalent bonds. Proteins are polymers of amino acids, polysaccharides of monosaccharides, nucleic acids of nucleotides.

The point every examiner picks up: lipids are not strictly macromolecules. Their molecular weight does not exceed 800 Da, so they should have gone into the acid-soluble pool. Why do they appear in the acid-insoluble fraction? Because in the cell lipids are arranged into membranes (cell membrane and the membranes of organelles). When a tissue is ground, the membranes are broken into pieces that form vesicles, and these vesicles are not water-soluble; they end up in the retentate along with the true macromolecules. So lipids are counted with the macromolecular fraction for the practical reason of how they behave on grinding, not because of their size. This is a ready-made Assertion-Reason item and a 2-mark question, and the phrase to write is 'lipids are present as membranes which, on grinding, form insoluble vesicles'.

NCERT closes the section with a sentence on what the two fractions represent: the acid-soluble pool roughly gives the composition of the cytoplasm, and the macromolecules come from the cytoplasm and the organelles. Everything in the rest of the chapter is about the acid-insoluble fraction, taken in the order proteins (9.4), polysaccharides (9.5), nucleic acids (9.6), protein structure (9.7) and then enzymes (9.8), which are proteins with a special job.

A protein is a polypeptide, a linear chain of amino acids linked by peptide bonds. Since there are twenty types of amino acids (alanine, cysteine, proline, tryptophan, lysine and so on) and a protein contains several of them in a particular sequence, a protein is a heteropolymer, not a homopolymer. A homopolymer has only one type of monomer repeating (cellulose is one); a heteropolymer has more than one type. Some amino acids are essential, meaning the body cannot synthesise them and they must come from the diet; the rest are non-essential. Dietary proteins are the source of essential amino acids, which is why the quality of a protein food is judged by its essential amino acid content.

Proteins do many things in living organisms. NCERT's Table 9.4 gives six examples that turn up as 1-mark matching items: collagen, the intercellular ground substance; trypsin, an enzyme; insulin, a hormone; antibody, which fights infectious agents; receptor, for sensory reception (smell, taste, hormone and so on); and GLUT-4, which enables glucose transport into cells. Two superlatives are pure MCQ fodder: collagen is the most abundant protein in the animal world, and RuBisCO (ribulose bisphosphate carboxylase-oxygenase) is the most abundant protein in the whole of the biosphere. Other functions worth one line: proteins transport nutrients across membranes, some fight infection, some are hormones, some are enzymes.

Exercise 9.5 tests whether you understand the primary structure: if a method tells you which amino acid is at each of the two ends (the N-terminal and the C-terminal), then a pure, homogeneous protein sample will give a single amino acid at each end; a mixture of proteins will show more than one N-terminal or C-terminal amino acid. Exercise 9.8, curdling of milk, is answered from the same section: the milk protein casein is denatured by the acid produced by the lactic acid bacteria, its globular tertiary structure is lost and the protein coagulates as curd.

Polysaccharides are the second class of macromolecules in the acid-insoluble fraction: long chains of sugars. The monomers are monosaccharides, linked by glycosidic bonds (a bond between two adjacent monosaccharides formed by dehydration, the −OH of one sugar joining a carbon of the next with loss of water). Exercise 9.2 asks you to illustrate it: draw two glucose rings joined through an oxygen bridge between C-1 of one and C-4 of the next and label it 'glycosidic bond'.

Cellulose is a polysaccharide made of only one type of monosaccharide, glucose; it is a homopolymer. Starch is a variant of cellulose, also made of glucose, and is the energy store of plants. Glycogen is the corresponding store in animals, a branched glucose polymer (NCERT's figure of a portion of glycogen shows the branch points). Inulin is a polymer of fructose. A polysaccharide chain has two different ends: the right end is the reducing end and the left end is the non-reducing end (the reducing end has a free anomeric carbon that can act as a reducing agent). In glycogen, because of branching, there is one reducing end and many non-reducing ends.

The iodine test comes up in every third paper. Starch forms helical secondary structures; the helix can hold iodine (I₂) molecules inside it, and this complex gives the blue colour. Cellulose does not contain complex helices, so it cannot hold iodine and gives no colour with it. Plant cell walls are made of cellulose; paper made from plant pulp and cotton fibre are cellulose. Then the complex polysaccharides: some have as building blocks amino sugars and chemically modified sugars, for example glucosamine and N-acetyl galactosamine. The exoskeletons of arthropods contain the complex polysaccharide chitin. NCERT states that these complex polysaccharides are mostly homopolymers. Exercise 9.12 asks what gums are made of: gums are heteropolysaccharides of several monosaccharide units (arabinose, galactose and so on), secreted by plants; Fevicol is different, a synthetic polymer (polyvinyl acetate based), not a polysaccharide.

The third class of macromolecules in the acid-insoluble fraction is the nucleic acids, DNA and RNA, which are polynucleotides. Together with polysaccharides and polypeptides they make up the true macromolecular fraction of any tissue. The building block is the nucleotide, which has three chemically distinct components: a heterocyclic compound (the nitrogen base), a monosaccharide (a pentose) and a phosphoric acid or phosphate. The heterocyclic compounds are the purines adenine and guanine and the pyrimidines uracil, cytosine and thymine. The sugar is a five-carbon ring: ribose (a monosaccharide pentose) in RNA and 2'-deoxyribose in DNA. The rules: uracil in RNA only, thymine in DNA only; adenine, guanine and cytosine in both.

In a polynucleotide the nucleotides are joined by phosphodiester bonds: the phosphate on C-5' of one nucleotide is esterified to the −OH on C-3' of the sugar of the next, so one phosphate forms two ester linkages, hence 'diester'. The result is a sugar-phosphate backbone with the bases sticking out. That is the third bond of exercise 9.2.

Nucleic acids have a secondary structure, and for DNA the Watson-Crick model is the answer to the standard 5-mark question. Its salient features, in the order a marking scheme rewards: (1) DNA is made of two polynucleotide strands; (2) the two strands are antiparallel, one running 5'→3' and the other 3'→5'; (3) the backbone of each strand is sugar-phosphate and the bases project inside towards the axis; (4) the bases of the two strands pair by hydrogen bonds, A with T (two H-bonds) and G with C (three H-bonds), so the strands are complementary; (5) the two strands are coiled into a right-handed double helix; (6) in the common B-DNA form the helix has a pitch of 34 Å (3.4 nm), with 10 base pairs per turn, so the rise per base pair is 3.4 Å (0.34 nm). NCERT mentions that other secondary structures of DNA also exist (A-DNA, C-DNA, Z-DNA). RNA is usually single-stranded but folds on itself into secondary structures (hairpins, as in tRNA). The exemplar short answer 'nucleic acids exhibit secondary structure, justify with examples' is answered by the double helix of DNA and the clover-leaf folding of tRNA.

Proteins, NCERT says, are heteropolymers containing strings of amino acids, and their structure is described at four levels. The primary structure is the sequence of amino acids in the polypeptide: the positional information, which amino acid is first, which second and so on. The first amino acid, with a free amino group, is the N-terminal amino acid; the last, with a free carboxyl group, is the C-terminal amino acid. The primary structure therefore has exactly two ends, and a chain is drawn as a line with the N-terminus on the left. The links are peptide bonds, formed when the carboxyl group of one amino acid reacts with the amino group of the next with loss of a water molecule (−CO−NH−); this is the second bond of exercise 9.2.

A protein thread does not exist as a straight line; it folds. The secondary structure is the regular, repeated local folding of the backbone. The thread is folded in the form of a helix, and only right-handed helices are observed in proteins; the α-helix is the example. Other regions of the protein thread are folded into other forms, such as the β-pleated sheet. Secondary structure is held by hydrogen bonds between backbone groups.

In the tertiary structure the long protein chain, already helical in parts, is folded upon itself like a hollow woollen ball, giving a three-dimensional view of the protein. NCERT stresses that the tertiary structure is absolutely necessary for the many biological activities of proteins: the active site of an enzyme, for instance, is a pocket created by this folding (9.8). This is the answer to exercise 9.3.

Some proteins are an assembly of more than one polypeptide, and the arrangement of these individual folded polypeptides or subunits with respect to each other (linear string of spheres, spheres arranged one upon another in a cube or plate, and so on) is the quaternary structure. The set-piece example is adult human haemoglobin, which consists of four subunits, two identical α subunits and two identical β subunits. Learn the four levels with one line each and one example (insulin sequence for primary, α-helix for secondary, myoglobin or an enzyme for tertiary, haemoglobin for quaternary); that is the whole 3-mark or 5-mark answer.

The rationalised NCERT has removed the separate section on the nature of the bonds linking monomers, but exercise 9.2 still asks you to illustrate the three bonds, and each is described within 9.4 to 9.6. Take them together so that one answer serves all questions. In a polysaccharide the monomers are monosaccharides linked by a glycosidic bond. It is formed by dehydration: the −OH group on one carbon (usually C-1) of a sugar reacts with an −OH on another carbon (usually C-4) of the next sugar, a molecule of water is lost and an oxygen bridge C−O−C is left between the two rings. To illustrate, draw two hexagonal glucose rings side by side, join them through a single O between C-1 of the first and C-4 of the second, and write 'glycosidic bond' with an arrow at the bridge oxygen.

In a polypeptide the amino acids are linked by peptide bonds. It is also formed by dehydration: the carboxyl group (−COOH) of one amino acid reacts with the amino group (−NH₂) of the next, water is lost and the linkage −CO−NH− results. To illustrate, write H₂N−CH(R₁)−CO−NH−CH(R₂)−COOH and box the −CO−NH−; the free amino end is the N-terminal, the free carboxyl end the C-terminal.

In a nucleic acid the nucleotides are linked by phosphodiester bonds. The phosphate group forms an ester bond with the C-3' hydroxyl of the sugar of one nucleotide and another ester bond with the C-5' hydroxyl of the sugar of the next, so the phosphate is esterified twice, a diester, and the chain has a sugar-phosphate backbone. To illustrate, draw two pentose rings one below the other, a phosphate between them connected to C-3' of the upper and C-5' of the lower sugar, and the base on C-1' of each. All three bonds are covalent and all three form by loss of water; that is the common thread the examiner likes in the concluding sentence. (Hydrogen bonds, by contrast, hold the two DNA strands and the α-helix together and are not the bonds between monomers.)

Almost all enzymes are proteins. The exception you must state: some nucleic acids that behave like enzymes are called ribozymes. An enzyme, like any protein, has a primary structure (the amino acid sequence) and a secondary and tertiary structure. When you look at the tertiary structure you notice that the backbone of the protein chain folds upon itself, the chain criss-crosses itself, and hence many crevices or pockets are made. These pockets are the active sites. An enzyme by its very nature is a protein with a three-dimensional structure, including an active site, and the substrate fits into that site. In this sense enzymes are catalysts like inorganic catalysts (they speed up a reaction without being consumed), but with two differences: enzymes are far more specific, and enzymes are sensitive to temperature in a way inorganic catalysts are not.

That sensitivity is the property called thermolability. Inorganic catalysts work efficiently at high temperatures and high pressures; enzymes get damaged at high temperatures, NCERT says above about 40 °C, because the tertiary structure that makes the active site is disrupted (denaturation). The exception, again for the Assertion-Reason item: enzymes isolated from organisms that normally live at extremely high temperatures, the thermophilic organisms, retain their catalytic power even at 80 to 90 °C. Thermal stability is therefore an important quality of enzymes isolated from thermophilic organisms, used for example in industrial processes.

Exercise 9.15, 'describe the important properties of enzymes', is answered by collecting the points of this and the next sections: enzymes are mostly proteins (ribozymes excepted); they have an active site; they are highly specific for their substrate (one enzyme, one reaction); they are required in very small amounts and are not consumed in the reaction; they lower the activation energy and greatly increase the rate (carbonic anhydrase, ten million times); they are thermolabile and pH-sensitive with an optimum of each; their activity depends on substrate concentration up to V_max; they are inhibited by specific chemicals (competitive inhibition); many need co-factors. Keep this list ready as a 5-mark answer.

NCERT distinguishes a physical change (a change in shape without breaking bonds, such as ice melting or water evaporating) from a chemical reaction, in which bonds are broken and made and a new compound results. Its example of an inorganic chemical reaction is Ba(OH)₂ + H₂SO₄ → BaSO₄ + 2H₂O. Hydrolysis of starch to glucose is an organic chemical reaction. The rate of a reaction is the amount of product formed per unit time, δP/δt, and rates depend on temperature: a general rule of thumb NCERT states is that the rate doubles or decreases by half for every 10 °C change in either direction. Enzyme-catalysed reactions have rates vastly higher than uncatalysed ones. The example to quote with numbers: carbonic anhydrase, which catalyses CO₂ + H₂O → H₂CO₃. In the absence of the enzyme about 200 molecules of H₂CO₃ form per hour; with carbonic anhydrase about 600,000 molecules form per second, an acceleration of about 10 million times.

Most reactions in the body are multistep. Glucose is converted to pyruvic acid in ten steps in glycolysis, each step catalysed by a different enzyme; such a series of reactions is a metabolic pathway. A metabolic pathway can branch, and the same glucose can end differently: in skeletal muscle under anaerobic conditions it gives lactic acid, under aerobic conditions pyruvic acid, and in yeast during fermentation ethanol. The reactants and products of these reactions are the metabolites.

Now the mechanism, section 9.8.2. For a substrate S to become a product P, S must pass through a higher-energy state called the transition state; the difference between the average energy of S and the energy of the transition state is the activation energy. Picture the energy diagram: energy on the y-axis, progress of reaction on the x-axis; the curve rises from S to a peak (the transition state) and then falls to P. If P is at a lower energy level than S the reaction is exothermic and spontaneous, though it may still be slow because of the barrier; if P is at a higher level the reaction is endothermic and needs energy. In either case the peak has to be crossed. Enzymes lower the activation energy: by binding the substrate at the active site and straining its bonds, they provide an alternative path through a transition state of lower energy, so many more substrate molecules can cross the barrier per unit time. The enzyme itself is unchanged at the end. In the diagram draw two curves from S to P, the higher peak labelled 'without enzyme' and the lower 'with enzyme', and mark the activation energy for each with a vertical arrow from S's level to the peak.

Each enzyme has a substrate-binding site in its molecule, so a highly reactive enzyme-substrate complex (ES) is formed. This complex is short-lived and dissociates into the product P and the unchanged enzyme, with an intermediate enzyme-product complex (EP) in between. The overall scheme, which you must write as the first line of the answer, is E + S → ES → EP → E + P. The formation of the ES complex is essential for catalysis; without it the enzyme cannot lower the activation energy.

NCERT describes the catalytic cycle in four steps, and a 5-mark question wants all four with a diagram. Step 1: the substrate binds to the active site of the enzyme, fitting into it (the fit is often compared with a lock and key, the substrate being the key that fits only that enzyme's active site). Step 2: the binding of the substrate induces the enzyme to alter its shape, fitting more tightly around the substrate; this is the induced-fit idea. Step 3: the active site of the enzyme, now in close proximity to the substrate, breaks the chemical bonds of the substrate and the new enzyme-product complex is formed. Step 4: the enzyme releases the products of the reaction and the free enzyme is ready to bind another molecule of the substrate and run through the cycle again.

Three consequences to state. Because the enzyme is released unchanged, a small amount of enzyme can convert a large amount of substrate: enzymes are needed in tiny quantities and are not used up. Because the active site has a definite shape and chemistry, each enzyme acts on one substrate or one class of substrates: enzymes are specific. And because the active site is made by the tertiary structure, anything that disturbs that structure (heat, extreme pH) stops the enzyme. For the diagram, draw the enzyme as a large shape with a notch (the active site), the substrate as a piece that fits the notch, then the ES complex with the enzyme slightly changed in shape around the substrate, then EP with the substrate split into two product pieces, and finally the free enzyme and the released products, arranged in a circle with arrows.

The activity of an enzyme can be affected by a change in the conditions that alter the tertiary structure of the protein. These include temperature, pH, change in substrate concentration and the binding of specific chemicals that regulate its activity.

Temperature and pH. Enzymes generally function in a narrow range of temperature and pH. Each enzyme shows its highest activity at a particular temperature and pH called the optimum temperature and optimum pH; activity declines both below and above the optimum. Draw both graphs as bell-shaped curves with activity on the y-axis. The reasons differ on the two sides of the temperature curve and the examiner wants both: low temperature preserves the enzyme in a temporarily inactive state (the reaction is simply slow; the enzyme is not destroyed and recovers on warming), whereas high temperature destroys enzymatic activity because the protein is denatured (tertiary structure and active site lost). Human enzymes have an optimum around 37 °C; pepsin works at pH 2 and trypsin at about pH 8, which is why the same food is digested by different enzymes in the stomach and the intestine.

Concentration of substrate. With the increase in substrate concentration the velocity of the enzymatic reaction rises at first. The reaction ultimately reaches a maximum velocity (V_max), which is not exceeded by any further rise in substrate concentration. The reason: at high substrate concentration there are no free enzyme molecules left to bind with the additional substrate; every active site is occupied and the enzyme is saturated. Draw the graph as a curve rising steeply from the origin and flattening to a plateau labelled V_max; substrate concentration on the x-axis, velocity on the y-axis. (The substrate concentration at half V_max is the Michaelis constant K_m; NCERT does not use the term, so it is not required, but it does no harm to mark it.)

Inhibitors. The activity of an enzyme is also sensitive to the presence of specific chemicals that bind to it. When the binding of the chemical shuts off enzyme activity, the process is called inhibition and the chemical is an inhibitor. In competitive inhibition the inhibitor closely resembles the substrate in its molecular structure and competes with the substrate for the substrate-binding site (the active site). Because of this competition the substrate cannot bind, and the enzyme action declines. NCERT's example, which is compulsory in the answer: the inhibition of succinic dehydrogenase by malonate, which closely resembles the substrate succinate in structure. Competitive inhibition can be overcome by raising the substrate concentration, since the two are competing for the same site. Such competitive inhibitors are often used in the control of bacterial pathogens: a drug that mimics a bacterial substrate blocks the bacterial enzyme (sulpha drugs act this way).

Thousands of enzymes have been discovered, isolated and studied. Most of them have been classified into different groups based on the type of reaction they catalyse. Enzymes are divided into six classes, each with 4 to 13 subclasses, and each enzyme is named with a four-digit number (the EC number: class, subclass, sub-subclass and serial number). The six classes, in NCERT's order and words, are the substance of a standard 3-mark or 5-mark question, 'name the classes of enzymes and explain any two with the reaction type they catalyse'.

1. Oxidoreductases (dehydrogenases): enzymes that catalyse oxidoreduction between two substrates S and S', for example S reduced + S' oxidised → S oxidised + S' reduced. Example: succinic dehydrogenase, the alcohol dehydrogenases, the NAD-linked dehydrogenases of respiration. 2. Transferases: enzymes catalysing the transfer of a group G (other than hydrogen) between a pair of substrates S and S', for example S−G + S' → S + S'−G. Example: transaminases (amino group transfer), kinases (phosphate transfer, as hexokinase in glycolysis). 3. Hydrolases: enzymes catalysing hydrolysis of ester, ether, peptide, glycosidic, C−C, C−halide or P−N bonds. Example: amylase, lipase, trypsin, peptidases. 4. Lyases: enzymes that catalyse the removal of groups from substrates by mechanisms other than hydrolysis, leaving double bonds: X−C−C−Y → X−Y + C=C. Example: decarboxylases, aldolase, fumarase. 5. Isomerases: all enzymes catalysing inter-conversion of optical, geometric or positional isomers. Example: phosphohexose isomerase (glucose-6-phosphate to fructose-6-phosphate), triose phosphate isomerase. 6. Ligases: enzymes catalysing the linking together of two compounds, for example enzymes that catalyse the joining of C−O, C−S, C−N, P−O bonds, using ATP. Example: DNA ligase, aminoacyl-tRNA synthetase.

The examiner's favourite MCQs from this list: which class joins two compounds (ligases); which class hydrolyses peptide bonds (hydrolases); which class removes a group leaving a double bond (lyases); which class inter-converts isomers (isomerases); which class transfers a group other than hydrogen (transferases); which class catalyses oxidoreduction (oxidoreductases or dehydrogenases). The exemplar 'complete the reaction A reduced + A' oxidised →' is answered A oxidised + A' reduced, class oxidoreductase. A mnemonic in the order NCERT gives them: O T H L I L ('Oh, The Hydrolases Love Isomerase Ligases').

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. In these instances the protein portion of the enzyme is called the apoenzyme, and the complete, active enzyme (apoenzyme + co-factor) is the holoenzyme. NCERT identifies three kinds of co-factors, and the 3-mark question is 'name the three kinds with one example of each'.

Prosthetic groups are organic compounds that are distinguished from other co-factors in that they are tightly bound to the apoenzyme. Example: in peroxidase and catalase, which catalyse the breakdown of hydrogen peroxide to water and oxygen (2H₂O₂ → 2H₂O + O₂), 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; they serve as co-factors in a number of different enzyme-catalysed reactions rather than staying with one enzyme. The essential chemical components of many co-enzymes are vitamins: the co-enzymes NAD and NADP contain the vitamin niacin. (FAD contains riboflavin and coenzyme A contains pantothenic acid, useful extras.) Metal ions: a number of enzymes require metal ions for their activity; the ion forms coordination bonds with side chains at the active site and at the same time one or more coordination bonds with the substrate. Example: zinc is a co-factor for the proteolytic enzyme carboxypeptidase. Magnesium for many kinases and iron in cytochromes are other examples.

The closing sentence of the chapter is a marking point on its own: the catalytic activity is lost when the co-factor is removed from the enzyme, which testifies that co-factors play a crucial role in the catalytic activity of the enzyme. The exemplar asks how a prosthetic group differs from a co-factor; answer that a prosthetic group is one kind of co-factor, specifically an organic co-factor tightly (often covalently) bound to the apoenzyme, whereas co-factors as a group also include loosely and transiently bound co-enzymes and inorganic metal ions.