Biochemistry

Introduction to Biochemistry

Biochemistry studies the chemical processes occurring within and relating to living organisms. It sits at the intersection of biology and chemistry, providing molecular explanations for cellular functions including energy production, DNA replication, protein synthesis, signalling, and defence against disease. Its tools include spectroscopy, X-ray crystallography, NMR, mass spectrometry, and computational modelling.

✦ Four Major Macromolecule Classes

Proteins: polymers of amino acids; structural, enzymatic, signalling, transport, immune functions.

Carbohydrates: polymers of sugars; energy storage (glycogen, starch), structural (cellulose, chitin), signalling.

Lipids: fatty acids, glycerol, sterols; membrane structure (phospholipid bilayer), energy storage, hormones.

Nucleic Acids: DNA (genetic information storage), RNA (transcription, translation, regulation).

Proteins: Structure and Function

Proteins are linear polymers of 20 standard amino acids linked by peptide bonds (—CO—NH—). The sequence (primary structure) is encoded in DNA. Four levels of structure:

① Primary Structure

Amino acid sequence. Determined by gene sequence. The unique sequence determines all higher-order structure (Anfinsen's dogma: sequence → structure → function).

② Secondary Structure

Local regular folding: α-helix (3.6 residues per turn, stabilised by backbone H-bonds) and β-sheet (H-bonds between extended strands). Predicted by Ramachandran diagram.

③ Tertiary Structure

Complete 3D folding of single polypeptide chain, stabilised by hydrophobic interactions (dominant), disulfide bridges, H-bonds, and ionic interactions. Determined by X-ray crystallography, NMR, or cryo-EM.

④ Quaternary Structure

Assembly of multiple polypeptide chains. Example: haemoglobin (4 subunits: 2α + 2β), enabling cooperative oxygen binding. Not all proteins have quaternary structure.

Protein misfolding underlies devastating diseases: Alzheimer's (amyloid-β plaques, tau tangles), Parkinson's (α-synuclein aggregates), prion diseases (misfolded PrP propagates its own misfolding). AlphaFold2 (DeepMind, 2020) revolutionised structural biology by predicting protein structure from sequence with near-experimental accuracy using deep learning — solving a 50-year-old "protein folding problem."

Enzymes and Kinetics

Enzymes are biological catalysts — mostly proteins, some RNA (ribozymes) — that accelerate reactions by lowering activation energy without being consumed. Key features: high specificity (lock-and-key or induced-fit model), rate acceleration (10⁶–10¹⁷ fold), regulation by inhibitors, activators, and post-translational modification.

The Michaelis-Menten equation describes enzyme kinetics:

At [S] ≪ K_m: rate is proportional to [S] (first-order). At [S] ≫ K_m: rate approaches V_max (zero-order, enzyme saturated). The double-reciprocal Lineweaver-Burk plot linearises the equation to determine K_m and V_max experimentally. Enzyme inhibition types:

v = V_max · [S] / (K_m + [S]) v = reaction rate V_max = maximum rate (at enzyme saturation) [S] = substrate concentration K_m = Michaelis constant = [S] at v = V_max/2 (reflects enzyme-substrate affinity)

Carbohydrates and Lipids

Carbohydrates : monosaccharides (glucose C₆H₁₂O₆, fructose, galactose) → disaccharides (sucrose = glucose + fructose; lactose = glucose + galactose) → polysaccharides (starch and glycogen for energy storage; cellulose for structural support in plant cell walls — indigestible by most animals due to β-1,4-glycosidic bonds; chitin in arthropod exoskeletons and fungal cell walls).

Lipids : the phospholipid bilayer is the universal membrane structure — amphipathic phospholipids spontaneously form bilayers in water (hydrophilic heads face water; hydrophobic tails face inward). Cholesterol modulates fluidity. Fatty acids with all C-C single bonds are saturated (solid at room temperature: butter, lard); unsaturated fats contain C=C double bonds (liquid: olive oil). Trans fats (partially hydrogenated oils) are cardiotoxic — banned in many countries. Steroids (cholesterol, testosterone, cortisol, bile acids) are lipids derived from a 4-ring structure and serve as hormones and emulsifying agents.

Nucleic Acids

DNA (deoxyribonucleic acid) stores genetic information in a double helix (Watson and Crick, 1953; structure based on X-ray data by Franklin and Wilkins). Complementary base pairing: A=T (2 H-bonds), G≡C (3 H-bonds). The central dogma of molecular biology:

The human genome contains ~3.2 billion base pairs encoding ~20,000 protein-coding genes (only ~2% of total DNA); the remainder includes regulatory regions, transposons, repetitive sequences, and non-coding RNAs. CRISPR-Cas9 (2012, Charpentier & Doudna — Nobel 2020) enables precise genomic editing by directing a nuclease to a specific DNA sequence.

DNA → (transcription) → mRNA → (translation) → Protein Additional paths: reverse transcription (RNA → DNA in retroviruses) RNA replication (RNA viruses), direct RNA regulatory roles (non-coding RNA)

Metabolism: Energy from Glucose

Cells extract energy from glucose through a coordinated series of metabolic pathways:

ATP synthase (the "molecular turbine") uses the proton gradient across the inner mitochondrial membrane (chemiosmosis — Mitchell's hypothesis, Nobel 1978) to rotate and synthesise ATP from ADP + Pᵢ. It generates ~100 ATP molecules per second per molecule; each human body turns over its own body weight in ATP daily. Fermentation produces ATP anaerobically: 2 ATP per glucose, making it 15× less efficient than aerobic respiration.

Glycolysis (cytoplasm): Glucose (6C) → 2 Pyruvate (3C) Net yield: 2 ATP + 2 NADH Pyruvate decarboxylation → Acetyl-CoA + CO₂ Krebs (Citric Acid) Cycle (mitochondrial matrix): 2 Acetyl-CoA → 4 CO₂ + 6 NADH + 2 FADH₂ + 2 ATP Oxidative Phosphorylation (inner mitochondrial membrane): 10 NADH + 2 FADH₂ → ~34 ATP (via electron transport chain + ATP synthase) Total: 1 glucose → ~38 ATP (theoretical) / ~30-32 ATP (realistic)

Frequently Asked Questions

Enzyme specificity arises from precise structural complementarity between the enzyme's active site and its substrate. The active site is a 3D pocket formed by specific amino acid residues; only substrates with the correct shape, charge distribution, and hydrogen-bonding capacity bind productively. The "induced fit" model (Koshland, 1958) refines the simpler "lock-and-key" model by showing the active site dynamically adjusts its shape upon substrate binding, optimising contacts. This specificity is so precise that enzymes can distinguish between stereoisomers — reacting with only the L-amino acid or D-sugar form. Specificity is maintained despite proteins being "soft" nanometre-scale machines subject to thermal fluctuations at physiological temperature.

ATP (adenosine triphosphate) is the universal cellular energy carrier. Its role as "energy currency" comes from the large negative free energy released when it is hydrolysed: ATP + H₂O → ADP + Pᵢ, ΔG° = −30.5 kJ/mol (more under cellular conditions: ~−50 to −60 kJ/mol). This energy release drives thermodynamically unfavourable reactions when ATP hydrolysis is coupled to them. Just as money standardises economic exchange, ATP standardises cellular energy exchange — regardless of whether energy comes from glucose, fat, light (in plants), or other sources, it is converted to ATP for cellular use. The human body contains only ~250 g of ATP at any moment but turns over its entire body weight worth (~70 kg) of ATP in a day.

Denaturation is the disruption of a protein's 3D structure (secondary, tertiary, or quaternary) without breaking peptide bonds (primary structure). Agents: heat (vibrations break non-covalent bonds), extreme pH (changes ionisation of amino acid side chains), organic solvents (denature hydrophobic core), detergents, urea or guanidinium chloride (disrupt H-bonds). Result: protein unfolds, loses function (enzyme activity, structural integrity, receptor binding). A cooked egg white is an irreversible denaturation example — albumin permanently aggregates. Many proteins can refold spontaneously (reversible denaturation) if the denaturing agent is removed, demonstrating that sequence determines structure. Heat treatment of pathogens and food preservation rely on irreversible protein denaturation.

DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) differ chemically and functionally: DNA contains deoxyribose sugar (missing one OH group vs RNA's ribose), uses thymine (T) instead of RNA's uracil (U), and is typically double-stranded, enabling stable long-term genetic information storage. RNA is single-stranded, shorter-lived, contains ribose and uracil, and exists in multiple functional forms: mRNA (messenger — carries genetic codes to ribosomes), tRNA (transfer — brings amino acids), rRNA (ribosomal — structural and catalytic component of ribosomes), and various non-coding RNAs (miRNA, siRNA, lncRNA — regulatory). The ribose 2'-OH group makes RNA more reactive and less stable than DNA — suitable for transient messages, not permanent archives.

Glycolysis (from Greek: "sweet splitting") is a 10-step pathway in the cytoplasm that converts one glucose (6C) into two pyruvate (3C), with a net yield of 2 ATP and 2 NADH: Steps 1-5 (investment phase): 2 ATP consumed to phosphorylate glucose and split it into two 3C units (glyceraldehyde-3-phosphate). Steps 6-10 (payoff phase): each 3C unit generates 2 ATP and 1 NADH via substrate-level phosphorylation. Key enzymes: hexokinase (step 1, regulated), phosphofructokinase (step 3, major rate-controlling step, allosterically inhibited by ATP), pyruvate kinase (step 10). Glycolysis provides energy anaerobically, requires no oxygen, and is preserved across virtually all life forms — evidence of its ancient evolutionary origin.

The Krebs (citric acid/TCA) cycle is a series of 8 enzymatic reactions in the mitochondrial matrix that oxidises acetyl-CoA (2-carbon unit) to CO₂, regenerating oxaloacetate for the next cycle. Per acetyl-CoA: produces 3 NADH, 1 FADH₂, 1 GTP (= ATP), releases 2 CO₂. The NADH and FADH₂ carry electrons to the electron transport chain in oxidative phosphorylation — where most ATP is made. The cycle does much more than generate energy: it provides biosynthetic precursors for amino acid synthesis, gluconeogenesis, lipid synthesis, and haem biosynthesis. The cycle was elucidated by Hans Krebs in 1937 (Nobel 1953). Key regulatory enzymes (isocitrate dehydrogenase, α-ketoglutarate dehydrogenase) are inhibited by NADH and ATP — feedback inhibition matching cycle rate to cellular energy needs.

Cholesterol is an essential structural component of animal cell membranes (~30% of membrane lipid molecules). Its rigid ring structure inserts between phospholipid tails, affecting membrane fluidity in two ways: at high temperatures, cholesterol restrains phospholipid movement, reducing fluidity; at low temperatures, cholesterol prevents phospholipids from packing too tightly and reducing fluidity — acting as a "fluidity buffer." This regulation maintains optimal membrane fluidity for protein function and transport. Cholesterol is also the precursor for bile acids (fat digestion), steroid hormones (testosterone, estrogen, cortisol), and vitamin D. Excess LDL cholesterol in the blood deposits in arterial walls as plaques (atherosclerosis), leading to cardiovascular disease — mitigated by statins (HMG-CoA reductase inhibitors).

Oxidative phosphorylation (OXPHOS) is the mitochondrial process that produces ~34 of the ~38 ATP from aerobic glucose oxidation. It involves two coupled systems: (1) Electron Transport Chain (ETC): NADH and FADH₂ donate electrons to protein complexes (I-IV) in the inner mitochondrial membrane. Electrons pass through redox centres with decreasing energy, ultimately reducing O₂ to H₂O at complex IV (cytochrome c oxidase). The released energy pumps protons (H⁺) from the matrix to the intermembrane space, creating an electrochemical gradient. (2) ATP synthase (Complex V): Protons flow back down their concentration gradient through ATP synthase, driving rotation of its c-ring, which powers conformational changes in the F₁ domain that synthesise ATP. This is chemiosmosis, proposed by Peter Mitchell (Nobel 1978).

PTMs are chemical modifications to proteins after translation that alter their activity, stability, localisation, or interactions. Common PTMs: Phosphorylation (addition of -PO₄ by kinases; removed by phosphatases — the most widespread regulatory PTM, involved in virtually every signalling pathway). Glycosylation (addition of sugar chains — important for protein folding, stability, cell-cell recognition; all antibodies are glycoproteins). Ubiquitination (marks proteins for degradation by the 26S proteasome — vital for protein quality control and cell cycle regulation). Acetylation (histone acetylation opens chromatin, increasing gene expression). Methylation (often epigenetic regulation of gene expression). Hundreds of PTM types exist; mass spectrometry is the key tool for detecting them comprehensively.

CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats) is a bacterial adaptive immune system repurposed as a genome editing tool. It uses a guide RNA to direct the Cas9 endonuclease to a specific 20-nucleotide DNA sequence, where Cas9 creates a double-strand break. The cell repairs this break by either non-homologous end joining (NHEJ — disrupts the gene) or homology-directed repair (HDR — inserts a new sequence). Compared to earlier tools (zinc-finger nucleases, TALENs), CRISPR is far cheaper, faster, easier to program, and more widely accessible. Medical applications: CRISPR-based treatment for sickle cell disease approved (Casgevy, 2023, first CRISPR therapy in humans); clinical trials for cancer, blindness, HIV, and genetic diseases ongoing. Agricultural applications: disease-resistant crops, improved yields.

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