Biochemistry

Introduction

The Organelles and Their Functions

Marker Enzymes

Subcellular Fractionation

Tissue homogenised in isotonic sucrose → Differential centrifugation at increasing speed → 600 × g → nuclei → 10,000 × g → mitochondria, lysosomes, peroxisomes → 100,000 × g → microsomes (fragmented ER) → Supernatant → cytosol

• Density gradient centrifugation separates organelles of similar size but different density
• Purity is judged by marker enzyme assay — the practical reason markers matter

Mitochondria in Detail

• Double membrane; the outer is permeable (porins), the inner impermeable and folded into cristae
• Inner membrane — electron transport chain, ATP synthase, carrier proteins; rich in cardiolipin
• Matrix — TCA cycle, β-oxidation, PDH complex, urea cycle enzymes
• Own circular DNA (16.5 kb), its own ribosomes (70S) and tRNAs
• Maternally inherited — sperm mitochondria are degraded after fertilisation
• Regarded as descended from an engulfed bacterium — the endosymbiont theory, supported by the double membrane, circular DNA and 70S ribosomes

Lysosomes

• Single membrane; interior kept at pH 5 by an H+-ATPase
• Contain about 50 acid hydrolases — proteases, lipases, nucleases, glycosidases, sulphatases
• Called "suicide bags"; rupture causes autolysis
• Enzymes are tagged with mannose-6-phosphate in the Golgi for delivery
• Functions — digestion of engulfed material, autophagy, turnover of organelles, bone remodelling by osteoclasts

Compartmentation of Metabolic Pathways

• Compartmentation permits opposing pathways to run at once — fatty acid synthesis in the cytosol while oxidation proceeds in the mitochondrion — and allows each to be regulated separately

Applied Aspects

• Lysosomal storage diseases — deficiency of a single hydrolase causes accumulation of its substrate: Tay–Sachs, Gaucher, Niemann–Pick, Hurler, Pompe
• I-cell disease — failure to add the mannose-6-phosphate tag, so enzymes are secreted instead of delivered; lysosomes are empty and plasma hydrolase levels are high
• Mitochondrial diseases are maternally inherited and strike high-energy tissues — melas, MERRF, Leber hereditary optic neuropathy
• Zellweger syndrome — absent peroxisomes; very long chain fatty acids accumulate
• Marker enzymes in plasma indicate the site of cell damage — a rise in mitochondrial ast suggests severe hepatocyte necrosis rather than mere membrane leak

Composition

Fluid Mosaic Model

• Proposed by Singer and Nicolson (1972)
• A lipid bilayer behaving as a two-dimensional fluid, in which proteins float like icebergs in a sea
• Lateral diffusion is rapid; transverse movement (flip-flop) is very slow and needs flippase enzymes
• Asymmetric — phosphatidylcholine and sphingomyelin predominate on the outer leaflet, phosphatidylserine and phosphatidylethanolamine on the inner; all carbohydrate faces outward

Factors Affecting Membrane Fluidity

• Cholesterol acts in both directions — it is a fluidity buffer, keeping the membrane within a workable range

Transport Across the Membrane

Distinguishing simple from facilitated diffusion

• Facilitated diffusion is saturable, shows substrate specificity, can be competitively inhibited, and follows Michaelis–Menten kinetics
• Simple diffusion is linear with concentration and never saturates

The Sodium–potassium Pump

3 Na+ out, 2 K+ IN per ATP hydrolysed → Creates the Na+ and K+ gradients → Generates the resting membrane potential (electrogenic) → The Na+ gradient powers secondary active transport → Maintains cell volume by countering the Donnan effect

• It consumes 20–30% of resting ATP, and far more in kidney and brain
• Inhibited by digoxin and ouabain — the rise in intracellular sodium reduces Na+/Ca2+ exchange, so calcium accumulates and cardiac contractility increases

Other Membrane Functions

• Selective permeability and maintenance of the internal environment
• Signal transduction — receptors for hormones and neurotransmitters
• Cell recognition and adhesion — glycocalyx, blood group antigens, MHC molecules
• Electrical excitability in nerve and muscle
• Site of many enzymes — adenylate cyclase, alkaline phosphatase

Endocytosis and Exocytosis

• Receptor-mediated endocytosis is defective in familial hypercholesterolaemia — the LDL receptor cannot bind or internalise its ligand

Applied Aspects

• Cystic fibrosis — a defective CFTR chloride channel → viscid secretions; the sweat chloride test is diagnostic
• Cholera toxin locks adenylate cyclase on → massive chloride and water secretion. ORS works because SGLT-1 remains intact and glucose drags sodium and water with it
• Hereditary spherocytosis — defective spectrin and ankyrin of the membrane skeleton → loss of the biconcave shape and splenic destruction
• Liposomes, built from the same bilayer principle, are used to deliver amphotericin B and doxorubicin with less toxicity
• Local anaesthetics and general anaesthetics act on membrane channels and lipids
• Membrane fluidity falls with cholesterol loading, which stiffens the red cell in liver disease and produces target cells

Definition

• General formula (CH2O)n for most, though deoxyribose and the amino sugars do not fit it
• They are the chief source of dietary energy, supplying 4 kcal/g and 55–70% of calories in the Indian diet

Classification

Monosaccharides

By number of carbons

By functional group

• Aldoses — carry an aldehyde group at C-1: glucose, galactose, ribose
• Ketoses — carry a ketone group at C-2: fructose, ribulose, sedoheptulose

Disaccharides

Polysaccharides

A. Homopolysaccharides — one kind of monosaccharide

B. Heteropolysaccharides — more than one kind of unit

• Glycosaminoglycans — hyaluronic acid, chondroitin sulphate, keratan sulphate, dermatan sulphate, heparan sulphate, heparin
• Each is a repeating amino sugar + uronic acid disaccharide, usually sulphated
• Agar and gum acacia are plant heteropolysaccharides

Derived Carbohydrates

Applied Aspects

• Lactose intolerance — lactase deficiency, very common in Indian adults; the undigested sugar causes osmotic diarrhoea and flatulence
• Cellulose cannot be digested because man has no β-glucosidase, yet it is nutritionally important as dietary fibre
• Inulin clearance is the gold standard for GFR, though creatinine clearance is used in practice
• Sialic acid on the RBC surface gives the negative charge that keeps cells apart; its loss with ageing marks the cell for splenic removal
• Sugar alcohols are used as sweeteners but cause osmotic diarrhoea in quantity, and sorbitol must be avoided in hereditary fructose intolerance
• Dextran is used as a plasma volume expander, and is also the polysaccharide of dental plaque formed by oral streptococci from sucrose

Introduction

• A chiral carbon carries four different groups
• Number of possible isomers = 2n, where n is the number of chiral carbons
• Glucose has 4 chiral carbons → 24 = 16 isomers; fructose has 3 → 8

Types of Isomerism

D and L Isomerism

• Determined by the –OH on the penultimate (highest-numbered chiral) carbon — C-5 in a hexose
• –OH on the right = D form; on the left = L form, by reference to D- and L-glyceraldehyde
• Almost all naturally occurring sugars are of the D series
• D and L refer to configuration, not to the direction of optical rotation — D-fructose is in fact laevorotatory, which is why it was once called laevulose

Epimers

• The clinical importance is that galactose must be epimerised to glucose before it can be used — failure at any step of this pathway causes galactosaemia

Ring Structure, Anomers and Mutarotation

Open-chain glucose → The C-5 hydroxyl attacks the C-1 aldehyde → Forms an intramolecular hemiacetal → Six-membered pyranose ring (five-membered = furanose) → C-1 becomes a new chiral centre — the anomeric carbon → Two forms: α (–OH below the plane) and β (above)

• The anomeric carbon is C-1 in an aldose and C-2 in a ketose
• β-D-glucose is the more stable and predominates (about 64%) at equilibrium, since all its bulky groups are equatorial
• Haworth projections represent the ring; the chair conformation is the true shape

Mutarotation

• α-D-glucose has a specific rotation of +112°, β of +19°; the equilibrium mixture settles at +52.7°
• It proves that the ring opens and closes freely — which is exactly why the anomeric carbon can act as a reducing group
• Accelerated by acid, alkali and the enzyme mutarotase

Biological Significance of Configuration

• Enzymes are absolutely stereospecific — hexokinase phosphorylates D-glucose but not L-glucose
• α versus β determines digestibility: man can hydrolyse the α-1,4 bonds of starch but not the β-1,4 bonds of cellulose — the same monosaccharide, entirely different nutritional fate
• Only β-D-glucose is a substrate for glucose oxidase, which is why strips contain mutarotase
• Blood group antigens differ only in their terminal sugars and linkages

Optical Activity and its Measurement

• A solution of a chiral compound rotates the plane of polarised light
• Dextrorotatory (+ or d) rotates it clockwise; laevorotatory (− or l) anticlockwise
• Measured with a polarimeter; expressed as specific rotation
• Racemic mixture — equal amounts of both, giving no net rotation
• Examples — D-glucose is dextrorotatory (+52.7°), D-fructose is laevorotatory (−92°)
• Invert sugar — hydrolysis of sucrose (+66°) gives an equimolar mixture of glucose and fructose whose net rotation is negative; the sign inverts, hence the name. Honey is largely invert sugar

Applied Aspects

• Galactosaemia arises from failure to convert galactose to glucose — the clinical consequence of an epimerisation pathway defect
• L-sugars are not metabolised and have been explored as non-caloric sweeteners
• Polarimetry was historically used to measure urine glucose, exploiting optical rotation
• Enzymes distinguish stereoisomers absolutely, which is why L-glucose passes through the body unmetabolised while D-glucose is the central fuel of life
• Thalidomide is the classical warning from stereochemistry outside carbohydrate chemistry — one enantiomer sedative, the other teratogenic
• Glucose oxidase strips contain mutarotase, because the enzyme acts only on the β anomer and the sample contains both
• Honey is sweeter than sucrose because it is largely invert sugar, and fructose is the sweetest natural sugar
• Osazone crystals distinguish sugars in the laboratory, but glucose, fructose and mannose give identical crystals since they differ only at carbons involved in the reaction

Definition

• They are non-reducing in practical terms, tasteless and mostly insoluble — ideal for storage and structure, since they exert almost no osmotic pressure

Classification

• Homopolysaccharides — one type of monosaccharide
• Heteropolysaccharides — two or more types

Starch

• The chief dietary carbohydrate, from cereals, potato and pulses
• Digested by salivary and pancreatic α-amylase, which act only on α-1,4 bonds, yielding maltose, isomaltose and limit dextrins
• α-Amylase cannot cleave α-1,6 bonds — these need isomaltase at the brush border

Glycogen

• The storage polysaccharide of animals; liver 100 g, muscle 400 g
• Similar to amylopectin but more highly branched — a branch every 8–12 residues
• Branching gives many non-reducing ends, so glucose can be added or removed rapidly at many points at once; it also increases solubility
• Gives a red-brown colour with iodine
• Built on a protein primer, glycogenin

Cellulose

• The most abundant organic molecule on earth; the structural polysaccharide of plant cell walls
• Unbranched chains of glucose in β-1,4 linkage
• The β linkage lets the chains lie flat and hydrogen-bond into rigid fibrils — hence its strength
• Man has no β-1,4-glucosidase (cellulase), so it is entirely indigestible; ruminants rely on symbiotic bacteria
• Nutritionally important as insoluble dietary fibre

Comparison of the Three Glucose Polymers

Other Important Polysaccharides

Digestion of Dietary Polysaccharides

Starch in the mouth → Salivary α-amylase (ptyalin) — acts on α-1,4 only → Inactivated by gastric acid → Pancreatic α-amylase in the duodenum → Maltose, isomaltose, maltotriose, limit dextrins → Brush border: maltase, isomaltase, sucrase, lactase → Glucose, galactose, fructose absorbed

• Amylase cannot cleave the α-1,6 branch points, which is why isomaltase is required
• Glucose and galactose are absorbed by SGLT-1 (active, sodium-coupled); fructose by GLUT-5 (facilitated)

Applied Aspects

• Dietary fibre — insoluble (cellulose, hemicellulose, lignin) adds bulk and prevents constipation; soluble (pectin, gum, mucilage) lowers cholesterol and slows glucose absorption
• Glycaemic index depends partly on the amylose:amylopectin ratio — more amylose means slower digestion and a lower index
• Resistant starch escapes digestion and is fermented by colonic bacteria to short-chain fatty acids, which nourish the colonocyte
• Glycogen storage diseases result from defects in the enzymes that build or break these branches — Andersen disease affects the branching enzyme, Cori disease the debranching enzyme
• Heparin remains a mainstay anticoagulant, acting by activating antithrombin III
• Iodine staining distinguishes the polysaccharides — a simple bench test still used to demonstrate starch digestion
• Bacterial dextran on the tooth surface forms the matrix of dental plaque, which is why sucrose is more cariogenic than other sugars

Definition

• They are grouped by solubility, not by structure — which is why the class is so chemically varied

Bloor’s Classification

Simple Lipids

• Triacylglycerol (triglyceride) — three fatty acids esterified to glycerol; the storage form of fat and about 95% of dietary lipid
• Fats are solid at room temperature (mostly saturated); oils are liquid (mostly unsaturated)
• Waxes — esters of fatty acids with long-chain monohydric alcohols; not digestible by man; found in cerumen and sebum

Compound Lipids

Derived Lipids

• Fatty acids — saturated and unsaturated
• Steroids — based on the cyclopentanoperhydrophenanthrene ring: cholesterol, bile acids, steroid hormones, vitamin D
• Ketone bodies — acetoacetate, β-hydroxybutyrate, acetone
• Fat-soluble vitamins — A, D, E, K
• Eicosanoids — prostaglandins, thromboxanes, leukotrienes

Functions of Lipids

Properties Used in the Laboratory

Amphipathic Lipids and Micelles

• Amphipathic = possessing both a polar (hydrophilic) and a non-polar (hydrophobic) region — phospholipids, cholesterol, bile salts, free fatty acids
• In water they orient with the polar heads outward, forming micelles, bilayers or liposomes
• Micelle formation is essential for fat digestion — bile salts emulsify fat and carry the products to the mucosal surface
• The lipid bilayer of every membrane is the direct consequence of this property
• Liposomes are used as drug delivery vehicles, as in liposomal amphotericin B and doxorubicin
• Neutral lipids such as triglyceride and cholesteryl ester are not amphipathic — they form the core of a lipoprotein, not its surface

Applied Aspects

• Trans fats, produced by partial hydrogenation, raise LDL and lower HDL — the worst combination; now legally restricted in India and many other countries
• Respiratory distress syndrome of the newborn follows deficiency of the phospholipid surfactant
• Lipid storage diseases — Gaucher, Niemann–Pick, Tay–Sachs and Krabbe disease all result from failure to degrade a complex lipid
• Cardiolipin is the antigen used in the VDRL test for syphilis, and antibodies to it explain the biological false positive in lupus
• Essential fatty acid deficiency causes scaly dermatitis and poor wound healing, seen in prolonged fat-free parenteral nutrition
• Steatorrhoea — failure to digest or absorb fat gives bulky, pale, offensive stools that float; caused by pancreatic insufficiency, bile salt deficiency or mucosal disease

Definition

• The even number arises because they are synthesised and degraded two carbons at a time
• Amphipathic — a polar carboxyl head and a non-polar hydrocarbon tail

Classification BY Chain Length

Classification BY Saturation

• Nomenclature — 18:2 Δ9,12 means 18 carbons, 2 double bonds, at positions 9 and 12 counting from the carboxyl end
• ω (omega) numbering counts from the methyl end — used nutritionally, because man cannot alter that end
• Naturally occurring unsaturated fatty acids are cis; the cis bond puts a kink in the chain, lowering the melting point and increasing membrane fluidity

Essential Fatty Acids

• Linoleic acid (ω-6) and α-linolenic acid (ω-3) are strictly essential
• Arachidonic acid is semi-essential — it can be made from linoleic acid
• Man lacks desaturases beyond carbon 9, so a double bond cannot be introduced at the ω-3 or ω-6 position
• Functions — eicosanoid precursors, membrane fluidity, retinal and brain development (DHA), lowering plasma cholesterol
• Deficiency — scaly dermatitis, poor wound healing, hair loss, fatty liver, growth retardation

Physical and Chemical Properties

Trans Fatty Acids

• Formed by partial hydrogenation of vegetable oils, as in vanaspati and margarine; also present naturally in small amounts in ruminant fat
• The trans configuration makes the chain straight, so it behaves like a saturated fat
• Raise LDL, lower HDL, raise Lp(a) and triglyceride — a worse profile than saturated fat, which raises LDL but does not lower HDL
• Also promote inflammation and insulin resistance
• Restricted by law in India to 2% of total fat, and banned or limited in many countries

Eicosanoid Precursors

• ω-3 and ω-6 compete for the same desaturases and elongases, and for cyclo-oxygenase — which is why the ratio matters more than the absolute intake of either
• This competition is the biochemical basis for the cardiovascular benefit attributed to fish oil

Applied Aspects

• Dietary recommendation — roughly equal parts saturated, monounsaturated and polyunsaturated fat, with an ω-6 : ω-3 ratio of 5:1 to 10:1. The modern Indian diet is often 15–20:1 because of heavy seed-oil use
• Fish oil (EPA and DHA) lowers triglyceride substantially and is used in severe hypertriglyceridaemia
• MCT oil bypasses the carnitine shuttle and needs no bile salts — valuable in fat malabsorption, chylothorax and carnitine deficiency
• DHA supplementation of infant formula supports retinal and neural development, since breast milk supplies it naturally
• Refrigeration and antioxidants delay rancidity — the more unsaturated the oil, the more readily it oxidises, which is why fish oil spoils fastest
• Peroxisomal disorders — Zellweger syndrome and adrenoleucodystrophy result from failure to oxidise very long chain fatty acids
• Rancid fat is harmful as well as unpalatable — the peroxides formed destroy vitamins A and E and generate free radicals
• Coconut oil is unusual among plant oils in being highly saturated, though largely medium-chain, which alters its metabolic handling
• Mustard oil is rich in erucic acid, which has been linked to myocardial lipidosis in animals; the evidence in man is not established

Definition and General Structure

• 20 standard amino acids are found in proteins
• All except glycine have a chiral α-carbon and therefore show optical isomerism
• All amino acids in human protein are of the L configuration
• Proline is an imino acid — its nitrogen is part of a ring

Classification BY Side Chain Polarity

Classification BY Nutritional Requirement

• Mnemonic — "MATT VILPHLy", or "Any Help In Learning These Little Molecules Proves Truly Valuable"
• Arginine and histidine are essential in growth but adequately synthesised by healthy adults

Classification BY Metabolic Fate

Properties

Zwitterion and isoelectric pH

• At physiological pH an amino acid exists as a zwitterion — the amino group is protonated (–NH3+) and the carboxyl group is ionised (–COO−)
• It is therefore amphoteric, acting as both acid and base — the basis of the buffering action of proteins
• Isoelectric pH (pI) = the pH at which the molecule carries no net charge and does not move in an electric field
• Solubility is minimum at the pI — used to precipitate proteins, as in casein preparation

Other properties

• Optical activity — all except glycine
• Ultraviolet absorption at 280 nm by the aromatic amino acids
• Ninhydrin reaction — gives a purple colour with all amino acids except proline, which gives yellow

Non-protein Amino Acids and Derivatives

Titration Curve and Buffering

• A simple amino acid such as glycine has two ionisable groups, and therefore two pK values — about 2.3 for –COOH and 9.6 for –NH3+
• The titration curve shows two buffering zones, one around each pK
• PI = the average of the two pK values flanking the zwitterion; for glycine, (2.3 + 9.6) ÷ 2 = 5.97
• Acidic amino acids have a low pI, basic ones a high pI
• Histidine is the only amino acid with a pK near physiological pH (imidazole, pK 6.0) — which is why it is the chief buffering residue of haemoglobin and of proteins generally
• Cysteine and lysine residues are the usual sites of covalent modification and of heavy metal binding

Applied Aspects

• Inborn errors of amino acid metabolism — phenylketonuria, alkaptonuria, maple syrup urine disease, homocystinuria; most are detected on newborn screening by tandem mass spectrometry
• Chromatography and electrophoresis separate amino acids by charge and polarity; the basis of amino acid profiling in metabolic disease
• Protein quality depends on the essential amino acid pattern — lysine is limiting in cereals, methionine in pulses, which is why cereal–pulse combinations complement each other
• D-amino acids occur in bacterial cell walls and some antibiotics; the reason penicillin can target bacteria selectively

Definition

The Peptide Bond

–COOH of one amino acid + –NH2 of the next → Loss of water (condensation) → peptide (amide) bond

• Partial double bond character from resonance → it is rigid and planar, and cannot rotate
• Almost always in the trans configuration — except before proline
• Rotation is possible only about the φ (phi) and ψ (psi) bonds either side of the α-carbon; the permitted angles are shown on a Ramachandran plot
• Uncharged but polar; participates in hydrogen bonding

Levels of Organisation

Primary structure

• Determined entirely by the gene
• It dictates all higher levels — a protein folds spontaneously into its native shape, as Anfinsen showed
• Insulin was the first protein sequenced (Sanger, 1955)
• A single change can be catastrophic — sickle cell disease is one amino acid substitution

Secondary Structure

Tertiary and Quaternary Structure

• Hydrophobic interactions are the chief driving force — non-polar side chains turn inward, away from water, while polar ones face outward
• Disulphide bonds between cysteine residues are the only covalent bonds stabilising tertiary structure; abundant in secreted proteins such as insulin and immunoglobulins
• Chaperones assist folding and prevent aggregation
• Domains — independently folding functional units
• Quaternary structure allows co-operativity and allosteric regulation — haemoglobin (α2β2), lactate dehydrogenase (tetramer), creatine kinase (dimer)

Denaturation

• Effects — loss of biological activity, decreased solubility, increased viscosity, greater susceptibility to proteases
• Sometimes reversible (renaturation) if mild, which is itself the proof that primary structure determines conformation

Forces Stabilising Protein Structure

• Only the peptide and disulphide bonds are covalent — all higher structure depends on weak forces acting in large numbers, which is exactly why proteins are so easily denatured

Applied Aspects

• Sickle cell disease — Glu→Val at position 6 of the β chain creates a hydrophobic patch, so deoxyhaemoglobin polymerises. A primary structure change causing a quaternary structure disease
• Prion diseases — Creutzfeldt–Jakob disease and kuru; a normal α-helical protein refolds into a β-sheet form that templates the same change in others. Disease from misfolding alone, with no change in sequence
• Amyloidosis and Alzheimer disease — β-sheet aggregates deposited in tissue; stain with Congo red giving apple-green birefringence
• Denaturation in practice — sterilisation by heat, disinfection by alcohol, and the use of egg white or milk as a first-aid antidote in heavy metal poisoning
• Osteogenesis imperfecta — a glycine substitution in collagen disrupts the triple helix, since only glycine is small enough for the interior position

Introduction

• Serum = plasma minus fibrinogen and the other clotting factors
• All plasma proteins except the γ-globulins are made in the liver

Separation BY Electrophoresis

• On cellulose acetate or agarose at pH 8.6, proteins carry a net negative charge and migrate to the anode
• Five bands are resolved: albumin, α1, α2, β, γ

Albumin

• The most abundant plasma protein; 585 amino acids, 69 kDa; half-life 20 days
• Contributes 75–80% of the plasma oncotic pressure, because of its high concentration and small size
• Transport — free fatty acids, unconjugated bilirubin, calcium, thyroxine, steroid hormones, and many drugs
• Buffering; a reserve of amino acids

Causes of hypoalbuminaemia

• Reduced synthesis — chronic liver disease, malnutrition (kwashiorkor)
• Increased loss — nephrotic syndrome, protein-losing enteropathy, extensive burns
• Increased catabolism — sepsis, malignancy, trauma
• Dilution — overhydration, pregnancy
• Consequence — oedema and ascites; also altered free drug levels, since less is protein-bound

Important Individual Proteins

Acute Phase Response

• Driven by IL-6, IL-1 and TNF-α acting on the liver
• The liver reprioritises — making defence proteins at the expense of transport proteins. This is why albumin falls in any acute illness and is a poor marker of nutrition

Characteristic Electrophoretic Patterns

Applied Aspects

• Serum albumin is a poor marker of acute nutrition — its half-life is 20 days and it falls in any inflammatory state. Prealbumin (2 days) and retinol-binding protein (12 hours) respond faster
• Corrected calcium must be calculated when albumin is low, since about half of plasma calcium is albumin-bound: add 0.8 mg/dL for every 1 g/dL the albumin is below 4
• Drug displacement — in hypoalbuminaemia the free fraction of phenytoin or warfarin rises, causing toxicity at a normal total level
• Bence Jones protein — free light chains in urine in myeloma; they precipitate at 45–60 °C and redissolve on boiling

Definition

• Exception — ribozymes are catalytic RNA molecules (peptidyl transferase of the ribosome)
• Accelerate reactions 103 to 1017 fold
• Highly specific and can be regulated — the two features that distinguish them from chemical catalysts

Terminology

Iubmb Classification — Six Classes

• Mnemonic — "Over The Hill Like I Live"
• Each enzyme has a four-digit EC number; for example hexokinase is EC 2.7.1.1

Mechanism of Enzyme Action

E + S → Enzyme–substrate (ES) complex → Transition state stabilised → E + P

How the rate is increased

• Lowering the activation energy — the fundamental mechanism. The enzyme provides an alternative path with a lower energy barrier
• Proximity and orientation — substrates are held close together in the correct alignment
• Strain and distortion of the substrate bond
• Acid–base catalysis — by histidine, aspartate, glutamate residues
• Covalent catalysis — a transient covalent bond with the enzyme, as in chymotrypsin

Theories of Enzyme–substrate Binding

Enzyme Specificity

Factors Affecting Enzyme Activity

• Substrate concentration — hyperbolic curve; plateaus at Vmax
• Enzyme concentration — linear relation, the basis of enzyme assays
• Temperature — optimum 37 °C in man; activity doubles for every 10 °C rise (Q10 = 2) until denaturation above 50–60 °C
• PH — each enzyme has an optimum: pepsin 1–2, salivary amylase 6.8, trypsin 8, alkaline phosphatase 9–10
• Product concentration — accumulation slows the reaction
• Activators and inhibitors

Applied Aspects

• Enzymes are named by adding "-ase" to the substrate (urease) or the reaction (dehydrogenase); older trivial names persist — pepsin, trypsin, ptyalin
• Fever raises enzyme activity, contributing to the increased metabolic rate; hypothermia lowers it, which is why it protects the brain during cardiac surgery
• Acidosis and alkalosis shift enzymes away from their optimum pH and impair metabolism
• Inborn errors of metabolism are almost all single enzyme defects — phenylketonuria, galactosaemia, glycogen storage diseases
• Many drugs act as enzyme inhibitors — aspirin on cyclo-oxygenase, statins on HMG-CoA reductase, allopurinol on xanthine oxidase

Effect of Substrate Concentration

• At low [S] the reaction is first order — rate is proportional to [S]
• At high [S] it becomes zero order — rate is independent of [S], because all active sites are saturated
• The curve is a rectangular hyperbola

Michaelis–menten Equation

Assumptions

• Only the initial velocity is measured, so the reverse reaction is negligible
• A steady state exists — ES is formed and broken down at equal rates
• [S] is far greater than [E]

Significance of KM

• Km is a measure of the affinity of the enzyme for its substrate
• Low Km = high affinity (saturated at low substrate)
• High Km = low affinity
• It is a constant for a given enzyme and substrate, independent of enzyme concentration
• Units are those of concentration (mol/L)
• Helps identify which isoenzyme is present, and predicts behaviour at physiological substrate levels

Lineweaver–burk Plot

• Advantage — gives accurate values of Km and Vmax without needing very high substrate concentrations, and distinguishes types of inhibition at a glance
• Other plots — Eadie–Hofstee, Hanes–Woolf

Allosteric Enzymes Do Not Obey Michaelis–menten

• They give a sigmoid curve, not hyperbolic
• Because of co-operativity between subunits — binding at one site alters affinity at the others
• The measure used is K0.5 or S0.5, not Km
• Examples — phosphofructokinase-1, aspartate transcarbamoylase; haemoglobin behaves the same way though it is not an enzyme

Enzyme Units

Measurement of Enzyme Activity

• Enzymes are measured by their activity, not their mass — they are present in far too small an amount to weigh
• Conditions must be standardised — optimum pH, 37 °C, saturating substrate, so that the rate depends on enzyme concentration alone
• Zero-order conditions are used deliberately: with substrate in excess, velocity is proportional to the amount of enzyme
• Only the initial velocity is measured, before product accumulates
• Coupled assays are used when the product is not directly measurable — the reaction is linked to one that changes NADH absorbance at 340 nm

Applied Aspects

• Glucokinase mutations cause MODY-2 (maturity onset diabetes of the young) — a raised Km resets the glucose sensor of the β cell to a higher threshold
• Alcohol dehydrogenase has a low Km and is saturated at low blood alcohol → alcohol is cleared by zero-order kinetics, at a constant rate irrespective of concentration
• Phenytoin, aspirin and ethanol all show zero-order elimination at therapeutic doses → small dose increases cause large rises in plasma level and toxicity
• Knowing Km guides the design of competitive inhibitors as drugs
• Enzyme deficiency states may be due to a raised Km rather than an absent enzyme — some respond to large doses of the vitamin coenzyme, as in vitamin-responsive homocystinuria

Definition and Classification

• Reversible — binds non-covalently; can be removed by dialysis
• Irreversible — binds covalently; the enzyme is permanently inactivated

Competitive Inhibition

• The inhibitor is structurally similar to the substrate
• It competes for the same active site
• The effect can be overcome by increasing substrate concentration

Examples

• Malonate inhibits succinate dehydrogenase — the classical example
• Methanol poisoning treated with ethanol — ethanol competes for alcohol dehydrogenase and prevents formation of toxic formaldehyde and formate
• Sulphonamides compete with PABA for bacterial dihydropteroate synthase
• Statins compete with HMG-CoA for HMG-CoA reductase
• Allopurinol competes with hypoxanthine for xanthine oxidase
• Methotrexate competes with folate for dihydrofolate reductase

Non-competitive Inhibition

• The inhibitor binds at a site other than the active site (allosteric)
• It binds equally to E and ES
• Not overcome by increasing substrate

• Examples — heavy metals (lead, mercury, silver) binding sulphydryl groups; cyanide on cytochrome oxidase; fluoride on enolase

Uncompetitive Inhibition

• The inhibitor binds only to the ES complex, never to free enzyme
• Both Km and Vmax are decreased, in the same proportion
• Lineweaver–Burk gives parallel lines
• Example — lithium on inositol monophosphatase

Comparison Table

Irreversible and Suicide Inhibition

• Irreversible — covalent modification of the active site
• Organophosphates phosphorylate the serine of acetylcholinesterase
• Aspirin irreversibly acetylates cyclo-oxygenase
• Penicillin irreversibly inhibits bacterial transpeptidase
• Iodoacetate inhibits glyceraldehyde-3-phosphate dehydrogenase

Suicide (mechanism-based) inhibition

• The inhibitor is a substrate analogue that the enzyme itself converts into a reactive species, which then destroys the enzyme
• Examples — allopurinol on xanthine oxidase (also competitive), 5-fluorouracil on thymidylate synthase, α-methyldopa
• Advantage — very high specificity, since only the target enzyme activates it

Applied Aspects

• Antimetabolites in cancer therapy — methotrexate, 5-fluorouracil, 6-mercaptopurine all work as enzyme inhibitors
• Ethanol as an antidote in methanol and ethylene glycol poisoning is the classic clinical use of competitive inhibition; fomepizole is the modern alternative
• Heavy metal poisoning treated with chelating agents — BAL, EDTA, penicillamine — which remove the metal from the sulphydryl groups
• Feedback inhibition by the end product is usually allosteric and is the commonest physiological form of regulation

WHY Regulation Is Needed

Mechanisms — Overview

Allosteric Regulation

• The regulator binds at a site distinct from the active site (Greek allos = other)
• Positive modulator → increases activity; negative modulator → decreases it
• Allosteric enzymes are oligomeric and show sigmoid kinetics
• Feedback (end-product) inhibition is the commonest form — the final product of a pathway inhibits the first committed enzyme

Covalent Modification

• The commonest is phosphorylation and dephosphorylation
• Protein kinases phosphorylate (using ATP) at serine, threonine or tyrosine; phosphatases remove it
• Ultimately controlled by hormones through second messengers

Glucagon / adrenaline → ↑ cAMP → Protein kinase A → Phosphorylates target enzymes → Glycogen phosphorylase activated; glycogen synthase inactivated

Zymogens (proenzymes)

• Inactive precursors activated by irreversible proteolytic cleavage
• Purpose — prevents the enzyme digesting the cell that makes it, and allows a rapid, amplifying response

Induction and Repression

• Alters the amount of enzyme by changing the rate of transcription; slow but long lasting
• Inducible enzymes — glucokinase, HMG-CoA reductase, ala synthase, the urea cycle enzymes, cytochrome P450
• Insulin induces glucokinase and the lipogenic enzymes; glucagon and cortisol induce the gluconeogenic enzymes
• Constitutive enzymes are produced at a constant rate regardless of demand

Applied Aspects

• Acute pancreatitis — premature intrapancreatic activation of trypsinogen → autodigestion; the whole point of the zymogen system is to prevent this
• Enzyme induction by drugs — rifampicin, phenytoin, carbamazepine and alcohol induce cytochrome P450 → failure of oral contraceptives and warfarin
• Enzyme inhibition by drugs — cimetidine, erythromycin, ketoconazole inhibit P450 → toxicity of co-administered drugs
• α1-antitrypsin deficiency — loss of a natural protease inhibitor → unopposed elastase → emphysema and liver disease
• Phenobarbitone induces UDP-glucuronyl transferase — formerly used to treat neonatal jaundice by increasing bilirubin conjugation

Principle

Cardiac Markers

• Troponin has replaced all others for diagnosis; the others retain value only for timing

Liver Enzymes

Other Diagnostic Enzymes

Isoenzymes and Their Diagnostic Value

Lactate dehydrogenase — 5 isoenzymes

• A tetramer of H (heart) and M (muscle) subunits
• LDH-1 (H4) — heart and RBC
• LDH-2 (H3M) — reticuloendothelial system; normally the highest in serum
• LDH-3 — lung; LDH-4 — kidney, placenta
• LDH-5 (M4) — liver and skeletal muscle
• "Flipped pattern" LDH-1 > LDH-2 indicates myocardial infarction

Creatine kinase — 3 isoenzymes

• CK-MM — skeletal muscle; CK-MB — heart; CK-BB — brain

Factors to Consider Before Interpreting a Raised Enzyme

• Tissue specificity — how confined is the enzyme to one organ?
• Timing — when did the injury occur relative to the sample?
• Magnitude — a rise of 10× means something different from 1.5×
• Half-life — determines how long the abnormality persists
• Route of clearance — renal or hepatic failure may raise levels without tissue damage
• Physiological causes — ALP rises in growing children, in pregnancy, and after a fatty meal (intestinal isoenzyme)
• Artefacts — haemolysis of the sample falsely raises LDH, ast and potassium

Applied Aspects

• Enzymes are used therapeutically too — streptokinase and urokinase as thrombolytics, asparaginase in leukaemia, pancreatin in pancreatic insufficiency
• Enzymes as laboratory reagents — glucose oxidase for blood glucose, urease for urea, and horseradish peroxidase in ELISA
• Timing matters — a normal troponin within 4 hours of chest pain does not exclude infarction; the test must be repeated
• The pattern of enzymes is more informative than any single value — ALT with ALP, or ast with ALT, localises the disease far better than either alone
• Serial measurement beats a single reading — a falling trend indicates recovery, a rising one continuing damage
• Normal enzymes do not exclude disease — in established cirrhosis the transaminases may be normal because little viable liver remains

Definition

Reactions

A. Energy investment phase (2 ATP used)

Glucose → Hexokinase / glucokinase — ATP used, irreversible → Glucose-6-phosphate → Phosphohexose isomerase → Fructose-6-phosphate → phosphofructokinase-1 — ATP used, rate-limiting, irreversible → Fructose-1,6-bisphosphate → Aldolase → Glyceraldehyde-3-phosphate + DHAP

• DHAP is converted to G3P by triose phosphate isomerase, so all six carbons continue — hence everything after this point is × 2

B. Energy generation phase (4 ATP made)

Glyceraldehyde-3-phosphate → G3P dehydrogenase — NADH produced; inhibited by iodoacetate → 1,3-bisphosphoglycerate → Phosphoglycerate kinase — ATP made (substrate-level) → 3-phosphoglycerate → Phosphoglycerate mutase → 2-phosphoglycerate → Enolase — inhibited by fluoride → Phosphoenolpyruvate → pyruvate kinase — ATP made, irreversible → pyruvate

The Three Irreversible Steps

• These three are bypassed by separate enzymes in gluconeogenesis

Energetics

• Substrate-level phosphorylation occurs at two steps — phosphoglycerate kinase and pyruvate kinase. These need no oxygen, which is why RBC survive

Regulation

• Fructose-2,6-bisphosphate is the most important physiological activator of PFK-1. It is made by PFK-2, which insulin activates and glucagon inactivates — the link between hormone and pathway
• Citrate inhibits PFK-1 — when the TCA cycle is well supplied, glycolysis slows

Fate of Pyruvate

• The purpose of forming lactate is to regenerate NAD+, without which glycolysis would stop at the G3P dehydrogenase step

Significance

• The only source of ATP for RBC, cornea, lens and renal medulla
• Provides rapid ATP in exercising muscle when oxygen is limiting
• Provides intermediates — DHAP for glycerol-3-phosphate (triglyceride synthesis), 3-phosphoglycerate for serine
• Supplies 2,3-BPG in the RBC through the Rapoport–Luebering shunt
• It is the common pathway for all dietary hexoses — fructose and galactose enter it

Applied Aspects

• Pyruvate kinase deficiency — the second commonest cause of hereditary non-spherocytic haemolytic anaemia; the RBC cannot make enough ATP for its Na+–K+ pump
• Fluoride inhibits enolase — hence sodium fluoride is added to blood collection tubes for glucose estimation, to stop glycolysis in the sample
• Arsenic poisoning — arsenate substitutes for phosphate at the G3P dehydrogenase step, so glycolysis continues but no ATP is formed
• Warburg effect — tumour cells use aerobic glycolysis even with adequate oxygen; the basis of FDG-pet scanning
• Lactic acidosis — from tissue hypoxia, shock, or metformin overdose
• Hexokinase deficiency — a rare haemolytic anaemia; unlike pyruvate kinase deficiency, 2,3-BPG is low, so the anaemia is poorly tolerated
• Cancer chemotherapy increasingly targets glycolytic enzymes, exploiting the tumour cell's dependence on this pathway

Definition

Reactions

Acetyl-CoA (2C) + Oxaloacetate (4C) → Citrate synthase → Citrate (6C) → Aconitase → Isocitrate → isocitrate dehydrogenase — NADH + CO2; rate-limiting → α-Ketoglutarate (5C) → α-KG dehydrogenase — NADH + CO2 → Succinyl-CoA (4C) → Succinate thiokinase — GTP (substrate-level) → Succinate → Succinate dehydrogenase — FADH2 → Fumarate → Fumarase → Malate → Malate dehydrogenase — NADH → Oxaloacetate regenerated

Energetics

• 2 CO2 are released per turn — at the isocitrate dehydrogenase and α-KG dehydrogenase steps
• The carbons released are not those of the entering acetyl group; they come from oxaloacetate
• Complete oxidation of one glucose = 32 ATP (glycolysis 7 + 2 PDH × 2.5 = 5 + TCA 2 × 10 = 20). Older texts give 38

Regulation

• The cycle is governed chiefly by the availability of NAD+ and ADP — that is, by the energy demand of the cell (respiratory control)
• Ca2+ stimulates three enzymes — linking muscle contraction directly to increased ATP production

Amphibolic Role

Catabolic — the final common pathway

• Oxidises acetyl-CoA derived from carbohydrate, fat and protein

Anabolic — intermediates are drawn off

Anaplerotic Reactions

Inhibitors

Applied Aspects

• Fatty acids cannot be converted to glucose — the two carbons entering as acetyl-CoA are lost as CO2, so there is no net gain of oxaloacetate
• Thiamine deficiency blocks the TPP-dependent α-KG dehydrogenase → accumulation of pyruvate and lactate → the neurological and cardiac features of beri-beri
• In starvation and diabetes oxaloacetate is drained into gluconeogenesis, so acetyl-CoA cannot enter the cycle and is diverted to ketone bodies — "fat burns in the flame of carbohydrate"
• Succinate dehydrogenase mutations cause phaeochromocytoma and paraganglioma
• Fluoroacetate poisoning — converted to fluorocitrate, which blocks aconitase; citrate accumulates and the cycle stops
• Isocitrate dehydrogenase (IDH) mutations occur in gliomas and acute myeloid leukaemia and are now therapeutic targets
• The cycle cannot run without oxygen even though it uses none directly — because NADH must be reoxidised by the respiratory chain

Definition

Substrates

The Four Key Enzymes — Bypassing Glycolysis

The Oxaloacetate Shuttle

Pyruvate enters mitochondrion → Pyruvate carboxylase → Oxaloacetate → OAA cannot cross the inner mitochondrial membrane → Reduced to malate (malate dehydrogenase) → Malate crosses into the cytosol → Re-oxidised to OAA → PEPCK → PEP

• This shuttle also carries NADH into the cytosol, which is needed at the G3P dehydrogenase step of gluconeogenesis
• When lactate is the substrate, NADH is generated in the cytosol already, so aspartate may be used instead of malate

Regulation

Fructose-2,6-bisphosphate — the master switch

Glucagon → ↑ cAMP → protein kinase A → Phosphorylates the bifunctional enzyme PFK-2 / FBPase-2 → Kinase activity off, phosphatase ON → ↓ Fructose-2,6-bisphosphate → ↓ PFK-1 and ↑ fructose-1,6-bisphosphatase → glycolysis off, gluconeogenesis ON

• This reciprocal control prevents a futile cycle in which glucose would be simultaneously made and broken down, wasting ATP as heat

Significance

• Maintains blood glucose during fasting, once liver glycogen is exhausted after about 18–24 hours
• Supplies the brain (120 g glucose/day) and the RBC, which are obligate glucose users
• Clears lactate produced by muscle and RBC (Cori cycle), preventing acidosis
• Clears glycerol from fat breakdown and propionate

Comparison with Glycolysis

• The two are reciprocally regulated so that they never run at full speed together — that would be a futile cycle, wasting ATP as heat

Applied Aspects

• Von Gierke disease (type I glycogen storage disease) — glucose-6-phosphatase deficiency → neither glycogenolysis nor gluconeogenesis can release glucose → severe fasting hypoglycaemia, lactic acidosis, hyperuricaemia, hepatomegaly
• Alcohol causes hypoglycaemia — its oxidation raises the NADH:NAD+ ratio, driving pyruvate to lactate and oxaloacetate to malate, so both gluconeogenic substrates are depleted. Dangerous in a fasting drinker
• Metformin reduces hepatic gluconeogenesis — its chief action in type 2 diabetes
• Biotin deficiency impairs pyruvate carboxylase and hence gluconeogenesis
• Cortisol excess (Cushing) → excess gluconeogenesis → steroid diabetes
• Hereditary fructose intolerance — aldolase B deficiency; fructose-1-phosphate accumulates and inhibits gluconeogenesis → severe hypoglycaemia after fructose

Normal Values

• Blood glucose is held within narrow limits because the brain and RBC depend on it absolutely

Sources and Fates of Blood Glucose

Hormonal Regulation

A. Insulin — the only hypoglycaemic hormone

• Secreted by β cells in response to a rise in blood glucose
• ↑ Glucose uptake by GLUT-4 in muscle and adipose tissue
• ↑ Glycogenesis; ↓ glycogenolysis
• ↓ Gluconeogenesis (represses PEPCK)
• ↑ Glycolysis (induces glucokinase, PFK, pyruvate kinase)
• ↑ Lipogenesis; ↓ lipolysis and ketogenesis

B. The counter-regulatory hormones

Response in the Fed State

Meal → ↑ Blood glucose → ↑ Insulin, ↓ glucagon → Liver: ↑ glycogenesis, ↑ glycolysis, ↑ lipogenesis → Muscle: ↑ GLUT-4 → ↑ uptake and glycogen → Adipose: ↑ uptake, ↑ triglyceride synthesis → Blood glucose returns to normal in 2 hours

Response in Fasting and Starvation

• Liver glycogen (about 100 g) lasts only 18–24 hours
• Muscle glycogen cannot raise blood glucose — muscle lacks glucose-6-phosphatase; it can only be used locally, or exported as lactate for the Cori cycle
• In prolonged starvation the brain adapts to use ketone bodies, which spares protein and prolongs survival

Glucose Tolerance Test

• 75 g oral glucose after an overnight fast; samples at 0 and 2 hours
• Normal — fasting < 100, 2 h < 140 mg/dL
• Impaired glucose tolerance — 2 h between 140 and 199
• Diabetes — fasting ≥ 126 or 2 h ≥ 200 mg/dL
• Lag storage curve — a high peak with rapid fall; seen after gastrectomy and in hyperthyroidism

Applied Aspects

• Diabetes mellitus — insulin deficiency or resistance → the cells are "starving in the midst of plenty"; unrestrained gluconeogenesis and lipolysis
• Hypoglycaemia — commonest cause is insulin or sulfonylurea therapy. Also insulinoma, alcohol, Addison disease, liver failure
• Whipple triad confirms true hypoglycaemia — symptoms, a low measured glucose, and relief on giving glucose
• HbA1c reflects the average glucose over 8–12 weeks — the standard measure of long-term control
• Dawn phenomenon — early-morning hyperglycaemia from the nocturnal surge of growth hormone and cortisol
• Somogyi effect — nocturnal hypoglycaemia provokes a counter-regulatory surge and rebound morning hyperglycaemia; treated by reducing, not increasing, the evening insulin
• Glycosuria appears above the renal threshold of 180 mg/dL; in pregnancy the threshold falls, giving benign glycosuria

Definition and Importance

• Site — mitochondrial matrix
• It is irreversible — the single most important consequence being that fatty acids can never be converted to glucose
• It is the gateway linking glycolysis to the TCA cycle

Components

Steps

Decarboxylation — pyruvate + TPP → hydroxyethyl-TPP + CO2 → Oxidation — transferred to lipoamide as acetyl group → Transfer — acetyl group to CoA → acetyl-CoA → Reoxidation — reduced lipoamide → FAD → FADH2 → NAD+ → NADH

Regulation

A. Covalent modification — the chief mechanism

B. Product inhibition

• Acetyl-CoA and NADH inhibit the complex directly
• This is why β-oxidation of fatty acids switches off PDH — it floods the cell with acetyl-CoA and NADH, sparing glucose

Metabolic Significance

• Commits carbohydrate carbon irreversibly to oxidation or fat synthesis
• Its irreversibility is why acetyl-CoA is not glucogenic
• Ca2+ activation couples muscle contraction to fuel oxidation
• In the fed state insulin activates it → glucose is oxidised or converted to fat
• In fasting it is switched off → pyruvate is diverted to gluconeogenesis instead of being wasted

Related Complexes

• α-Ketoglutarate dehydrogenase of the TCA cycle and the branched-chain α-keto acid dehydrogenase use the same five coenzymes and the same mechanism
• Hence thiamine deficiency affects all three, and arsenite inhibits all three by binding lipoamide

Energetics and Position in Metabolism

• It is the only route by which carbohydrate carbon can enter the TCA cycle
• Because it is irreversible, carbohydrate can make fat but fat cannot make carbohydrate — one of the most consequential facts in metabolism

Fate of Acetyl-coa

• Acetyl-CoA is the central junction of metabolism — every major fuel converges on it, and several biosynthetic routes leave from it

Applied Aspects

• Thiamine deficiency → PDH cannot function → pyruvate and lactate accumulate → lactic acidosis, and the brain is deprived of acetyl-CoA. This underlies Wernicke encephalopathy and beri-beri
• Give thiamine before glucose in an alcoholic or malnourished patient — a glucose load consumes the last thiamine and can precipitate Wernicke encephalopathy
• PDH deficiency (inherited) — congenital lactic acidosis with neurological damage; treated with a ketogenic diet, which bypasses the block
• Arsenic poisoning — arsenite binds the sulphydryl groups of lipoamide → inhibits PDH; treated with BAL (dimercaprol)
• Dichloroacetate inhibits PDH kinase, keeping the complex active; used experimentally in congenital lactic acidosis
• Leigh syndrome — a mitochondrial encephalopathy in which PDH deficiency is one recognised cause