Organic Chemistry-5 (Molecules of Life) BCH6T15

Syllabus topics (RTMNU)

• Definition, classification of carbohydrates
• Preparation of glucose from Sucrose & Starch
• Reactions of glucose
• Mechanism of osazone formation
• Determination of open chain & cyclic structure of glucose
• Determination of ring size of monosaccharides
• Epimerisation, mutarotation
• Conversion of glucose into fructose and vice-versa
• Chain lengthening and shortening of aldoses (Wohl's method)
• Introduction to structures of maltose, sucrose, lactose, starch, cellulose, ribose and deoxyribose without involving structure determination

Carbohydrates: Definition and Classification

Carbohydrates are optically active polyhydroxy aldehydes or polyhydroxy ketones, or compounds that give these on hydrolysis. Many have the general formula C_x(H₂O)_y ("hydrates of carbon"), but this is not a strict rule (deoxyribose C₅H₁₀O₄ is a carbohydrate; acetic acid C₂H₄O₂ is not).

1. Monosaccharides: simplest sugars that cannot be hydrolysed further. By functional group — aldoses (glucose, ribose) and ketoses (fructose); by carbon number — trioses, tetroses, pentoses, hexoses (e.g. aldohexose glucose, ketohexose fructose).
2. Oligosaccharides: give 2–10 monosaccharide units on hydrolysis — disaccharides (sucrose, maltose, lactose), trisaccharides (raffinose).
3. Polysaccharides: give many units — homopolysaccharides (starch, cellulose, glycogen) and heteropolysaccharides (hyaluronic acid).

Also: reducing sugars (reduce Tollens'/Fehling's; free aldehyde/ketone or hemiacetal group — glucose, maltose, lactose) and non-reducing sugars (sucrose); sugars (sweet, crystalline, water-soluble) and non-sugars (starch, cellulose).

Preparation of Glucose from Sucrose and Starch

Reactions of Glucose

Mechanism of Osazone Formation

Glucose reacts with three molecules of phenylhydrazine on warming:

1. Phenylhydrazone formation: C-1 aldehyde condenses with one PhNHNH₂ → –CH=N–NHPh + H₂O.
2. Oxidation of C-2: the phenylhydrazone tautomerises (to an enamine/keto form); loss of aniline gives an imine at C-1 and a carbonyl at C-2 — overall, the C-2 –CHOH– is oxidised to C=O while one PhNHNH₂ is reduced to aniline and ammonia.
3. Second condensation: the new C-2 carbonyl reacts with the third PhNHNH₂, and the C-1 imine exchanges with phenylhydrazine (releasing NH₃), giving the osazone: HC=N–NHPh at C-1 and C=N–NHPh at C-2.

Open Chain and Cyclic Structure of Glucose

Open chain (Fischer): from the reactions above — molecular formula C₆H₁₂O₆; straight chain of 6 C (HI); one –CHO (HCN, NH₂OH, Br₂ water); five –OH (pentaacetate); one –CH₂OH (HNO₃ gives a dicarboxylic acid with 6 C). Hence CH₂OH–(CHOH)₄–CHO, with D-configuration (–OH of C-5 on the right).

Objections to the open chain: glucose does not give Schiff's test or a bisulphite compound; glucose pentaacetate does not react with NH₂OH (no free –CHO); glucose exists in two crystalline forms — α (m.p. 146 °C, [α]_D +112°) and β (m.p. 150 °C, [α]_D +18.7°); it shows mutarotation; and it forms two methyl glucosides.

Cyclic structure: the C-5 –OH adds to the C-1 aldehyde to form a six-membered hemiacetal ring (pyranose). C-1 becomes a new chiral centre (the anomeric carbon), giving α-D-glucopyranose (C-1 –OH on the right in Fischer / down in Haworth) and β-D-glucopyranose (up in Haworth). These two are anomers.

Determination of Ring Size of Monosaccharides

1. Methylation (Haworth) method: glucose → methyl glucoside (CH₃OH/HCl) → exhaustive methylation (dimethyl sulphate/NaOH) → methyl tetra-O-methylglucoside → hydrolysis of the glycosidic methyl → 2,3,4,6-tetra-O-methyl-D-glucose. The free –OH at C-5 shows C-5 was in the ring. Oxidation with HNO₃ gives trimethoxyglutaric acid (from C-1 to C-5), confirming a 1,5 (pyranose) ring.

2. Periodate oxidation: methyl glucopyranoside consumes 2 mol HIO₄ and gives 1 mol formic acid (C-3 is lost as HCOOH between two cleavages). A furanoside would consume 2 mol HIO₄ but give formaldehyde (from C-6) and no formic acid. Thus glucose is a pyranose.

Epimerisation, Mutarotation and Interconversion of Glucose and Fructose

Epimers are diastereomers differing in configuration at only one chiral carbon — D-glucose and D-mannose (C-2), D-glucose and D-galactose (C-4). Epimerisation is the conversion of one into its epimer: e.g. D-gluconic acid heated with pyridine or quinoline partly changes into D-mannonic acid; this is converted to its lactone and reduced with Na–Hg to D-mannose. Dilute alkali also interconverts glucose, mannose and fructose through an enediol intermediate (Lobry de Bruyn–van Ekenstein rearrangement).

Mutarotation: the gradual change in specific rotation of a freshly prepared sugar solution until a constant value is reached. α-D-glucose (+112°) and β-D-glucose (+18.7°) both change to +52.7° — an equilibrium of about 36% α and 64% β, through a tiny amount of open-chain form. Catalysed by acids and bases.

Chain Lengthening and Shortening of Aldoses

Chain lengthening (Kiliani–Fischer synthesis): aldose + HCN → two epimeric cyanohydrins → hydrolysis → aldonic acids → lactones → reduction (Na–Hg) → two higher aldoses epimeric at C-2. Example: D-arabinose → D-glucose + D-mannose.

Chain shortening (Wohl's method): aldose + NH₂OH → oxime → heated with acetic anhydride and sodium acetate → dehydration to a nitrile with all –OH acetylated (penta-acetyl gluconitrile) → treated with sodium methoxide (or ammoniacal silver oxide) → removes acetyl groups and eliminates HCN → aldose with one carbon less. Example: D-glucose → D-arabinose.

Structures of Maltose, Sucrose, Lactose, Starch, Cellulose, Ribose and Deoxyribose

Likely exam questions

1. (Long, 8 marks) How is the open chain structure of glucose established? What are its limitations? Explain the cyclic structure of glucose.
2. (Long, 8 marks) Explain the mechanism of osazone formation. How is glucose converted into fructose and fructose into glucose?
3. (Short, 4 marks) What is mutarotation? Explain with reference to glucose.
4. (Short, 4 marks) Describe Wohl's method for chain shortening of aldoses.
5. (Short, 4 marks) How is the ring size of glucose determined by periodate oxidation?

Syllabus topics (RTMNU)

• Classification, structure and stereochemistry of amino acids
• Acid-base behavior, Zwitterion structure, isoelectric point and electrophoresis
• Determination of N-terminal amino acid (by DNFB and Edman method)
• Determination of C-terminal amino acid (by thiohydantoin and with carboxypeptidase enzyme)
• Structure and nomenclature of peptides and protein
• Classification of proteins
• Protein denaturation
• Structure determination of proteins (primary and secondary)

Amino Acids: Classification, Structure and Stereochemistry

α-Amino acids contain an amino group (–NH₂) and a carboxyl group (–COOH) on the same (α) carbon: R–CH(NH₂)–COOH. About 20 amino acids are found in proteins; they differ in the side chain R.

1. On the basis of R group: non-polar (glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, methionine); polar uncharged (serine, threonine, cysteine, tyrosine, asparagine, glutamine); acidic (aspartic acid, glutamic acid); basic (lysine, arginine, histidine).
2. On the basis of number of –NH₂ and –COOH: neutral (one each, e.g. glycine), acidic (two –COOH, e.g. glutamic acid), basic (two basic groups, e.g. lysine).
3. Nutritional: essential amino acids cannot be made by the body and must come from food — histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine; non-essential ones are made in the body.

Stereochemistry: except glycine (R = H), all α-amino acids have a chiral α-carbon and are optically active. Natural amino acids belong to the L-series (in the Fischer projection, with –COOH at the top, –NH₂ is on the left). By the CIP system most are S (cysteine is R because of sulphur). Isoleucine and threonine have two chiral centres.

Acid–Base Behaviour, Zwitterion and Isoelectric Point

In the solid state and in water an amino acid exists as a dipolar ion or zwitterion, H₃N⁺–CHR–COO⁻, formed by internal transfer of a proton from –COOH to –NH₂. This explains their high melting points (with decomposition), solubility in water, insolubility in non-polar solvents and large dipole moments. Amino acids are amphoteric:

Isoelectric point (pI): the pH at which an amino acid exists mainly as the zwitterion with zero net charge; it does not move in an electric field and has minimum solubility. For a neutral amino acid, pI = (pK_a1 + pK_a2) ÷ 2. Example: glycine, pK_a1 = 2.34, pK_a2 = 9.60 → pI = (2.34 + 9.60) ÷ 2 = 5.97. Acidic amino acids have low pI (glutamic acid ≈ 3.2); basic ones high pI (lysine ≈ 9.7).

Electrophoresis

Electrophoresis separates amino acids (or proteins) by their movement in an electric field on a buffer-soaked paper or gel at a fixed pH. At a pH below its pI an amino acid is positively charged and moves to the cathode; above its pI it is negative and moves to the anode; at its pI it does not move. Example: at pH 6.0, lysine (pI 9.7) moves to the cathode, glutamic acid (pI 3.2) to the anode, and glycine (pI ≈ 6.0) stays near the origin. Spots are located by spraying ninhydrin (purple colour).

Determination of N-Terminal Amino Acid

(a) Sanger's method (DNFB): the peptide is treated with 1-fluoro-2,4-dinitrobenzene (Sanger's reagent) in mild alkali (NaHCO₃). The free –NH₂ of the N-terminal residue displaces F (nucleophilic aromatic substitution) to give a yellow DNP-peptide. Complete acid hydrolysis then releases the DNP-amino acid (N-terminal) and free amino acids; the DNP-derivative is identified by chromatography. Drawback: the whole peptide is destroyed.

(b) Edman degradation: the peptide reacts with phenyl isothiocyanate (PhN=C=S) in mild base (pH about 8–9) to give a phenylthiocarbamoyl (PTC) peptide. Treatment with anhydrous acid (trifluoroacetic acid) cleaves only the N-terminal residue as a thiazolinone, which rearranges to a phenylthiohydantoin (PTH)-amino acid, identified by chromatography. The rest of the peptide remains intact and the cycle is repeated — the basis of automatic sequenators.

Determination of C-Terminal Amino Acid

(a) Thiohydantoin method: the peptide is heated with ammonium thiocyanate and acetic anhydride. The C-terminal carboxyl group cyclises with the adjacent amide to form a thiohydantoin of the C-terminal amino acid; this is cleaved off (by mild alkali) and identified, leaving the shortened peptide.

(b) Carboxypeptidase: the enzyme carboxypeptidase hydrolyses only the peptide bond of the C-terminal residue, releasing the C-terminal amino acid first. By measuring which amino acids appear with time, the C-terminal sequence of a few residues can be found.

Structure and Nomenclature of Peptides and Proteins

Amino acids join by peptide (amide) bonds (–CO–NH–) formed between the –COOH of one and the –NH₂ of the next, with loss of water. Two units give a dipeptide, three a tripeptide; many (>10) a polypeptide; above about 50–100 residues it is called a protein. The peptide bond is planar and rigid (partial double-bond character due to resonance) and usually trans.

Nomenclature: a peptide is written from the N-terminal (left) to the C-terminal (right). Residues other than the C-terminal take the suffix "-yl". Example: H₂N–CH₂–CO–NH–CH(CH₃)–COOH is glycylalanine (Gly-Ala); Ala-Gly-Phe is alanylglycylphenylalanine. Gly-Ala and Ala-Gly are different compounds.

Classification of Proteins

1. By shape: fibrous — long, insoluble, structural (keratin of hair, collagen, silk fibroin, myosin); globular — compact, soluble, functional (albumin, haemoglobin, enzymes, insulin).
2. By composition: simple — give only amino acids on hydrolysis (albumins, globulins); conjugated — protein + non-protein prosthetic group (glycoproteins, lipoproteins, nucleoproteins, chromoproteins such as haemoglobin, metalloproteins, phosphoproteins such as casein); derived — from partial hydrolysis (peptones, proteoses).
3. By function: enzymes (amylase), hormones (insulin), transport (haemoglobin), structural (collagen), defence (antibodies), storage (ovalbumin), contractile (actin, myosin).

Protein Denaturation

Denaturation is the loss of the natural three-dimensional (secondary, tertiary, quaternary) structure of a protein, and hence its biological activity, without breaking peptide bonds (primary structure stays). Causes: heat, strong acids or alkalis, urea, detergents, organic solvents (alcohol), heavy-metal ions, UV radiation. Hydrogen bonds, ionic and hydrophobic interactions are disrupted. Examples: coagulation of egg white on boiling, curdling of milk by lemon juice. Some proteins (e.g. ribonuclease, Anfinsen's experiment) renature when the agent is removed, showing that the primary structure decides the folding.

Structure Determination of Proteins: Primary and Secondary

Primary structure is the sequence of amino acids in the chain. First determined by F. Sanger for insulin (Nobel 1958). Steps:

1. Purify the protein; find molecular mass.
2. Break disulphide (–S–S–) bridges by performic acid oxidation or reduction with 2-mercaptoethanol.
3. Amino acid composition: complete hydrolysis (6 M HCl, 110 °C, about 24 h) and analysis in an amino acid analyser.
4. Identify N-terminal (DNFB, Edman) and C-terminal (carboxypeptidase) residues.
5. Partial hydrolysis into smaller fragments with specific reagents — trypsin (cuts after Lys, Arg), chymotrypsin (after Phe, Tyr, Trp), cyanogen bromide (after Met).
6. Sequence each fragment by Edman degradation and arrange them by overlapping sequences to get the full sequence.

Secondary structure is the regular local folding of the chain held by hydrogen bonds between C=O and N–H of peptide groups, proposed by Pauling and Corey (1951) and found by X-ray diffraction (also IR and CD spectra).

• α-Helix: right-handed coil; 3.6 residues per turn; pitch 0.54 nm; H-bond between C=O of residue n and N–H of residue n + 4, parallel to the axis; side chains point outward. Found in α-keratin (hair, wool).
• β-Pleated sheet: extended chains lie side by side, held by H-bonds between neighbouring chains; parallel or antiparallel; side chains above and below the pleated sheet. Found in silk fibroin.
• Others: β-turns and the triple helix of collagen.

Likely exam questions

1. (Long, 8 marks) Explain the zwitterion structure and isoelectric point of amino acids. How is electrophoresis used to separate amino acids?
2. (Long, 8 marks) Describe the methods for determination of N-terminal and C-terminal amino acids in a peptide.
3. (Short, 4 marks) Calculate the isoelectric point of glycine (pKa1 = 2.34, pKa2 = 9.60).
4. (Short, 4 marks) What is protein denaturation? Give two examples.
5. (Short, 4 marks) Describe the α-helix structure of proteins.

Syllabus topics (RTMNU)

• Components of nucleic acids: Adenine, guanine, thymine and Cytosine (Structure only)
• Other components of nucleic acids
• Nucleosides and nucleotides (nomenclature)
• Structure of polynucleotides
• Structure of DNA (Watson-Crick model) and RNA (types of RNA)
• Genetic Code
• Biological roles of DNA and RNA: Replication, Transcription and Translation

Components of Nucleic Acids: Bases

Nucleic acids (DNA and RNA) are polymers of nucleotides. Complete hydrolysis gives three components: a nitrogenous base, a pentose sugar and phosphoric acid.

Nitrogenous bases are of two types:

1. Purines (fused six- and five-membered rings, i.e. pyrimidine + imidazole): Adenine (A) = 6-aminopurine; Guanine (G) = 2-amino-6-oxopurine (2-amino-6-hydroxypurine in the enol form).
2. Pyrimidines (single six-membered ring with N at 1 and 3): Cytosine (C) = 4-amino-2-oxopyrimidine; Thymine (T) = 5-methyl-2,4-dioxopyrimidine (5-methyluracil), only in DNA; Uracil (U) = 2,4-dioxopyrimidine, only in RNA.

Other Components of Nucleic Acids

Pentose sugars: β-D-ribose in RNA and 2-deoxy-β-D-ribose (no –OH at C-2′) in DNA, both in the furanose form. Sugar carbons are numbered with primes (1′–5′) to distinguish them from base atoms.

Phosphoric acid (H₃PO₄): links sugars through phosphodiester bonds and gives nucleic acids their acidic nature (negative charge at physiological pH).

Nucleosides and Nucleotides: Nomenclature

Nucleoside = base + sugar, joined by a β-N-glycosidic bond from C-1′ of the sugar to N-9 of a purine or N-1 of a pyrimidine. Nucleotide = nucleoside + phosphate (usually esterified at C-5′, sometimes C-3′). Nucleotides are the monomers of nucleic acids.

Nucleotides with two or three phosphates — ADP, ATP, GTP — are energy carriers; ATP is the "energy currency" of the cell. Cyclic AMP is a second messenger.

Structure of Polynucleotides

Nucleotides are joined by 3′,5′-phosphodiester bonds: the phosphate on C-5′ of one sugar links to the –OH on C-3′ of the next. This forms a sugar–phosphate backbone with the bases projecting sideways. One end has a free 5′-phosphate (5′ end) and the other a free 3′-OH (3′ end); sequences are written 5′ → 3′, e.g. 5′-ATGC-3′. The order of bases (primary structure) carries the genetic information.

Structure of DNA: Watson–Crick Model

Proposed by J.D. Watson and F.H.C. Crick (1953) using Rosalind Franklin's X-ray data and Chargaff's rules (A = T, G = C).

1. Two polynucleotide strands coil around a common axis into a right-handed double helix.
2. The strands are antiparallel (5′→3′ and 3′→5′) and complementary.
3. Sugar–phosphate backbones are outside; bases are stacked inside, perpendicular to the axis.
4. Base pairing by hydrogen bonds: A=T (2 H-bonds), G≡C (3 H-bonds); a purine always pairs with a pyrimidine, so the width is uniform.
5. Diameter 2 nm; 10 base pairs per turn; one turn = 3.4 nm; 0.34 nm between base pairs; major and minor grooves present (B-DNA).

RNA: Types of RNA

1. mRNA (messenger RNA): copy of a gene; carries codons from DNA to ribosomes; short-lived.
2. tRNA (transfer RNA): small (about 75–90 nucleotides), clover-leaf shaped; carries a specific amino acid on its 3′-CCA end and reads the codon with its anticodon.
3. rRNA (ribosomal RNA): about 80% of cell RNA; forms ribosomes with proteins; 23S rRNA (prokaryotes) catalyses peptide bond formation (a ribozyme).
4. Small RNAs: snRNA (splicing), miRNA and siRNA (gene silencing).

Genetic Code

The genetic code is the relation between the base sequence of mRNA and the amino acid sequence of a protein. Its main features: triplet (3 bases = 1 codon; 64 codons); AUG is the start codon (methionine); UAA, UAG, UGA are stop codons; degenerate (most amino acids have more than one codon); non-overlapping and comma-less; unambiguous; nearly universal (exceptions in mitochondria); read 5′→3′; third base shows wobble. Deciphered by Nirenberg, Khorana and Holley (Nobel 1968).

Biological Roles of DNA and RNA: Replication, Transcription and Translation

Central dogma: DNA → (replication) DNA; DNA → (transcription) RNA → (translation) protein.

Replication — copying of DNA before cell division; semi-conservative (Meselson–Stahl, 1958): each new DNA has one old and one new strand. Helicase unwinds the helix at the origin; single-strand binding proteins and topoisomerase stabilise it; primase lays an RNA primer; DNA polymerase III (in E. coli) adds nucleotides 5′→3′ — continuously on the leading strand and as Okazaki fragments on the lagging strand; DNA polymerase I replaces primers; DNA ligase joins fragments.

Transcription — synthesis of RNA on a DNA template by RNA polymerase. The enzyme binds the promoter (in prokaryotes the −10 TATAAT and −35 TTGACA regions, recognised by the σ factor), unwinds DNA and makes RNA 5′→3′ complementary to the template strand (U opposite A), and stops at a terminator (rho-dependent or hairpin-type). In eukaryotes, pre-mRNA is processed: 5′ cap, 3′ poly-A tail and splicing out of introns.

Translation — synthesis of protein on ribosomes: (1) amino acids are activated and attached to tRNAs by aminoacyl-tRNA synthetases (using ATP); (2) initiation — ribosome assembles at the start codon AUG with initiator tRNA (fMet-tRNA in prokaryotes) in the P site; (3) elongation — aminoacyl-tRNA enters the A site, peptidyl transferase forms the peptide bond, and the ribosome moves one codon (translocation); (4) termination — at a stop codon, release factors free the polypeptide.

Likely exam questions

1. (Long, 8 marks) Describe the components of nucleic acids. Explain the Watson–Crick model of DNA.
2. (Long, 8 marks) Explain the biological roles of DNA and RNA in replication, transcription and translation.
3. (Short, 4 marks) Distinguish between a nucleoside and a nucleotide with examples.
4. (Short, 4 marks) Give the structures of adenine and thymine.
5. (Short, 4 marks) Write any four characteristics of the genetic code.

Syllabus topics (RTMNU)

• Enzyme: Introduction, Nomenclature and classification of Enzymes
• Fischer's lock and key and Koshland's induced fit hypothesis
• Michaelis Menten equation
• Identification of active site by the use of inhibitors
• Enzyme modification by site-directed mutagenesis
• Baker's yeast catalyzed reactions
• Vitamin: Introduction, classification of Vitamins
• Role of fat soluble Vitamin A, D, E, K and water soluble Vitamin B9, B12, and Vitamin C
• Dietary sources of Vitamins
• Diseases caused due to deficiency of Vitamin D, K, C and B12

Enzymes: Introduction

Enzymes are biological catalysts — mostly globular proteins (some RNA molecules, called ribozymes, also act as catalysts) — that speed up chemical reactions in living cells without being used up. They work under mild conditions (body temperature, near-neutral pH), are highly specific for their substrates, are needed in tiny amounts, and lower the activation energy. Many need a non-protein part: a cofactor (metal ion like Zn²⁺, Mg²⁺) or a coenzyme (organic, often from vitamins, e.g. NAD⁺, FAD). Protein part = apoenzyme; complete active enzyme = holoenzyme.

Nomenclature and Classification of Enzymes

Nomenclature: usually the suffix -ase is added to the name of the substrate (urease, maltase, lipase) or the reaction (oxidase, dehydrogenase, hydrolase). Some old trivial names are kept (pepsin, trypsin, rennin). The IUBMB system gives each enzyme a systematic name and a four-number EC number (e.g. EC 3.1.1.3 for lipase).

Fischer's Lock and Key and Koshland's Induced Fit Hypotheses

Lock and key model (Emil Fischer, 1894): the active site of the enzyme has a rigid shape exactly complementary to the substrate, like a lock to its key. Only the correct substrate fits, forming an enzyme–substrate (ES) complex, which changes into products. It explains specificity but not how one enzyme acts on several related substrates or how the transition state is stabilised.

Induced fit model (D.E. Koshland, 1958): the active site is flexible. When the substrate binds, the enzyme changes its shape to wrap around the substrate (like a glove on a hand), bringing catalytic groups into correct position and straining the substrate bonds. This explains group specificity, allosteric regulation and the action of inhibitors; supported by X-ray studies of hexokinase.

Michaelis–Menten Equation

Michaelis and Menten (1913) proposed that the enzyme (E) first combines reversibly with substrate (S) to form an ES complex, which breaks down to product (P) and free enzyme:

Meaning of K_m (Michaelis constant): the substrate concentration at which the rate is half of V_max. A low K_m means high affinity of the enzyme for its substrate. At low [S] (≪ K_m) the rate is first order in S; at high [S] (≫ K_m) the rate reaches V_max (zero order; enzyme saturated). The plot of v against [S] is a rectangular hyperbola. The Lineweaver–Burk double-reciprocal form, 1/v = (K_m/V_max)(1/[S]) + 1/V_max, gives a straight line with intercepts 1/V_max (y-axis) and −1/K_m (x-axis).

Numerical: an enzyme has V_max = 100 µmol min⁻¹ and K_m = 2 mM. At [S] = 2 mM, v = 100 × 2 ÷ (2 + 2) = 50 µmol min⁻¹ (half V_max). At [S] = 6 mM, v = 100 × 6 ÷ (2 + 6) = 75 µmol min⁻¹.

Identification of Active Site by the Use of Inhibitors

The active site is the small region of the enzyme where the substrate binds and catalysis occurs. Inhibitors help to find which amino acid residues are present there:

1. Irreversible (covalent) inhibitors react with a specific active-site residue and label it. Diisopropyl fluorophosphate (DIFP) reacts with only one serine (Ser-195) of chymotrypsin, out of many serines, and destroys activity — showing this serine is in the active site (serine proteases, acetylcholinesterase). Iodoacetamide labels essential cysteine residues.
2. Affinity labels (substrate-like reactive molecules): TPCK (tosyl-L-phenylalanine chloromethyl ketone) resembles chymotrypsin's substrate and alkylates histidine-57, proving His-57 is at the active site.
3. Competitive inhibitors (substrate analogues) bind at the active site and show its shape, e.g. malonate inhibits succinate dehydrogenase because it resembles succinate.

After labelling, the protein is hydrolysed and the labelled amino acid is identified and located in the sequence.

Enzyme Modification by Site-Directed Mutagenesis

Site-directed mutagenesis (developed by Michael Smith, Nobel 1993) changes a specific codon in the gene of an enzyme so that one chosen amino acid is replaced by another. Steps: (1) the gene is cloned; (2) a short mutagenic primer carrying the desired base change is used with DNA polymerase (now usually by PCR) to make the mutant gene; (3) the gene is expressed in bacteria to produce the modified enzyme; (4) its activity is tested.

Uses: (1) to confirm the role of active-site residues — e.g. replacing Ser-195 of a serine protease by alanine nearly abolishes activity; (2) protein engineering to make better enzymes — e.g. subtilisin used in detergents was made resistant to oxidation by replacing methionine-222; enzymes with higher heat stability or changed specificity.

Baker's Yeast Catalysed Reactions

Baker's yeast (Saccharomyces cerevisiae) is a cheap, easily available whole-cell biocatalyst containing many enzymes (dehydrogenases, decarboxylases). It works in water at room temperature and often gives optically active products.

1. Alcoholic fermentation: C₆H₁₂O₆ → (zymase) → 2 C₂H₅OH + 2 CO₂; sucrose is first hydrolysed by invertase.
2. Asymmetric reduction of ketones: ethyl acetoacetate is reduced by fermenting yeast (with sucrose) to (S)-ethyl 3-hydroxybutanoate with high enantiomeric excess: CH₃COCH₂COOC₂H₅ → CH₃CH(OH)CH₂COOC₂H₅.
3. Reduction of other carbonyls (β-keto esters, α-keto esters, diketones) and of activated C=C bonds.
4. Acyloin-type condensation: benzaldehyde added to fermenting yeast gives (R)-phenylacetylcarbinol (via pyruvate decarboxylase), an intermediate in the industrial synthesis of ephedrine.

Vitamins: Introduction and Classification

Vitamins are organic compounds needed in very small amounts in the diet for normal growth and health; the body cannot make them (or makes too little). The word "vitamine" was coined by Casimir Funk (1912). Many act as coenzymes.

Classification: (1) Fat-soluble — A, D, E, K: stored in liver and fat; excess may be toxic; (2) Water-soluble — B-complex (B₁, B₂, B₃, B₅, B₆, B₇, B₉, B₁₂) and C: not stored much (except B₁₂), excess passed in urine, needed regularly.

Role and Dietary Sources of Vitamins A, D, E, K, B9, B12 and C

Deficiency Diseases of Vitamins D, K, C and B12

1. Vitamin D: rickets in children (soft, bent bones — bow legs, knock knees, pigeon chest); osteomalacia in adults (softening and pain of bones).
2. Vitamin K: delayed blood clotting and haemorrhage (bleeding); newborns are given vitamin K to prevent bleeding disease.
3. Vitamin C: scurvy — bleeding and swollen gums, loose teeth, poor wound healing, joint pain, weakness.
4. Vitamin B₁₂: pernicious (megaloblastic) anaemia — large immature RBCs, tiredness; nerve damage (numbness, tingling), memory problems.

Likely exam questions

1. (Long, 8 marks) Derive the Michaelis–Menten equation and explain the significance of Km.
2. (Long, 8 marks) Describe the classification of vitamins. Give the role, dietary sources and deficiency diseases of vitamins D, K, C and B12.
3. (Short, 4 marks) Explain Koshland's induced fit hypothesis.
4. (Short, 4 marks) Write a note on baker's yeast catalysed reactions.
5. (Short, 4 marks) What is site-directed mutagenesis? Give one use.