Biomolecules: Carbohydrates, Lipids, Proteins, Enzymes and Nucleic Acids

Every structure a chiropractor examines, from the hydrated nucleus pulposus to the collagen of a ligament and the hemoglobin carrying oxygen to an injured muscle, is built from four families of molecules: carbohydrates, lipids, proteins and nucleic acids. The chemistry section of the board tests whether you know their structures well enough to predict function, and whether you can link a missing enzyme or a changed amino acid to a disease. This chapter builds that foundation and ties each molecule to the tissues and lab values you will meet in practice.

Section 1Carbohydrates

Monosaccharides

Carbohydrates have the general formula (CH2O)n. The simplest units, monosaccharides, are classified two ways: by the carbonyl group, as aldoses (an aldehyde at carbon 1) or ketoses (a ketone, usually at carbon 2), and by the number of carbons, as trioses, tetroses, pentoses and hexoses. Glyceraldehyde (an aldotriose) and dihydroxyacetone (a ketotriose) are intermediates of glycolysis. Ribose and deoxyribose are aldopentoses of RNA and DNA. The key hexoses are glucose (aldohexose, the main blood sugar), galactose (aldohexose, from lactose), mannose (aldohexose, used in glycoproteins) and fructose (ketohexose, from fruit and sucrose).

Stereochemistry and anomers

A carbon with four different substituents is a chiral center. Glucose in its open chain form has four chiral carbons (C2 to C5), giving 2⁴ = 16 stereoisomers. Several kinds of isomer are tested:

• Enantiomers are nonsuperimposable mirror images. The D or L label is set by the chiral carbon farthest from the carbonyl (C5 in glucose): hydroxyl on the right in the Fischer projection means D. Natural sugars are almost all D, while natural amino acids are L.
• Epimers differ in configuration at only one chiral carbon: glucose and galactose are C4 epimers; glucose and mannose are C2 epimers.
• Anomers differ only at the new chiral carbon created when the sugar cyclizes, the anomeric carbon (C1 in aldoses, C2 in ketoses).

In solution, hexoses exist mostly as rings. The C5 hydroxyl of glucose attacks the C1 aldehyde to form a cyclic hemiacetal, a six membered pyranose ring (fructose forms a five membered furanose through a hemiketal). Ring closure makes C1 chiral. In the Haworth projection of D-glucose, the α anomer has the C1 hydroxyl below the ring (trans to the CH2OH group at C5) and the β anomer has it above (cis). The two interconvert through the open chain in a process called mutarotation; at equilibrium about two thirds is β and one third α, with a trace of open chain. β-D-glucose is the most stable because in its chair form every bulky group is equatorial.

A sugar with a free anomeric carbon can open to an aldehyde and reduce copper or other oxidizing agents; it is a reducing sugar. All monosaccharides are reducing. This chemistry underlies the old Benedict test, and the same open chain aldehyde reacts nonenzymatically with protein amino groups in glycation, the basis of HbA1c and of advanced glycation end products that stiffen collagen in diabetes and aging.

Glycosidic bonds and disaccharides

A glycosidic bond forms when the anomeric hydroxyl of one sugar condenses with a hydroxyl of another, releasing water. The bond is named by the anomeric configuration and the carbons joined (for example α-1,4). Once the anomeric carbon is locked in a glycosidic bond it can no longer open, so a disaccharide is reducing only if one anomeric carbon remains free.

Polysaccharides

Starch, the plant storage form, has two parts: amylose, an unbranched chain of glucose in α-1,4 linkage that coils into a helix, and amylopectin, which adds α-1,6 branch points about every 24 to 30 residues. Glycogen, the animal storage form in liver and muscle, has the same bonds but branches more often, about every 8 to 12 residues. Heavy branching makes glycogen compact and soluble and gives many nonreducing ends for glycogen phosphorylase to attack at once, so glucose can be released fast. Cellulose, a structural plant polymer, is glucose in β-1,4 linkage; the straight chains hydrogen bond into fibers. Humans lack cellulase, so cellulose is dietary fiber, adding bulk and feeding colonic bacteria. Chitin (in fungal walls and arthropod exoskeletons) is a β-1,4 polymer of N-acetylglucosamine.

Glycoproteins, glycosaminoglycans and proteoglycans

Glycoproteins are proteins with short, branched oligosaccharide chains attached by N-linkage (to asparagine, begun in the rough ER) or O-linkage (to serine or threonine, in the Golgi). They are mostly protein by weight and include most plasma proteins, antibodies, hormones such as FSH and hCG, membrane receptors and the ABO blood group antigens.

Glycosaminoglycans (GAGs) are long, unbranched polymers of repeating disaccharides, typically an amino sugar (N-acetylglucosamine or N-acetylgalactosamine) plus a uronic acid (glucuronic or iduronic acid). Carboxyl and sulfate groups make them strongly negatively charged. The charges repel one another, so GAGs extend into stiff, space filling chains, and they attract cations, whose osmotic effect draws in large amounts of water. The result is a hydrated, compression resistant gel.

Proteoglycans are a core protein with many GAG chains attached, and unlike glycoproteins they are mostly carbohydrate (often 95 percent). The major proteoglycan of cartilage and disc is aggrecan: about 100 chondroitin sulfate and keratan sulfate chains on a core protein, many of which attach noncovalently, with the help of a link protein, to a central strand of hyaluronic acid. The resulting aggregate, trapped in a meshwork of collagen, behaves like a sponge in a net: proteoglycans pull water in and swell; collagen resists the swelling, generating the internal pressure that lets cartilage and the nucleus pulposus resist load. Under compression, water is squeezed out and returns when the load is removed, which is why the disc loses height during the day and regains it overnight. Smaller proteoglycans (decorin, biglycan) bind collagen and regulate fibril size.

Lysosomal enzymes degrade GAGs stepwise. Missing enzymes cause the mucopolysaccharidoses, in which GAGs accumulate and dermatan and heparan sulfate spill into urine: Hurler syndrome (α-L-iduronidase; corneal clouding, coarse facies, skeletal dysplasia) and Hunter syndrome (iduronate sulfatase; X-linked, no corneal clouding). Morquio syndrome affects keratan sulfate breakdown and produces severe skeletal dysplasia with odontoid hypoplasia and atlantoaxial instability.

Section 2Lipids

Lipids are a chemically diverse group defined by a shared property: they are poorly soluble in water and soluble in nonpolar solvents. They store energy, form membranes, insulate, and serve as hormones and signals.

Fatty acids

A fatty acid is a hydrocarbon chain with a carboxyl group at one end. Most natural fatty acids have an even number of carbons (12 to 24, most commonly 16 and 18) because they are built two carbons at a time. At physiological pH the carboxyl is ionized, so the molecule is amphipathic. Saturated fatty acids (palmitic, 16:0; stearic, 18:0) have no double bonds and pack tightly, so they are solid at room temperature. Unsaturated fatty acids have one (monounsaturated, oleic acid, 18:1 Δ9) or more (polyunsaturated) double bonds, almost always in the cis configuration, which puts a kink in the chain, lowers the melting point and increases membrane fluidity. Trans fats, made mainly by partial hydrogenation, are straight like saturated chains; they raise LDL, lower HDL and increase cardiovascular risk.

Two naming systems are used. The Δ system counts from the carboxyl carbon (C1). The omega (ω) system counts from the methyl end, so linoleic acid is an ω-6 fatty acid and α-linolenic acid an ω-3. Humans cannot insert double bonds beyond carbon 9, so linoleic and α-linolenic acids are essential. Linoleic acid is the precursor of arachidonic acid (20:4, ω-6), and α-linolenic acid gives rise to EPA and DHA (ω-3), important in retina and brain and with mildly anti-inflammatory effects.

Triglycerides

A triglyceride (triacylglycerol) is three fatty acids joined by ester bonds to glycerol. Triglycerides are neutral, very hydrophobic and stored nearly water free in adipocytes, which is why fat yields about 9 kcal/g compared with about 4 kcal/g for carbohydrate and protein, and why it is the body's main long term energy store. Hormone sensitive lipase in adipose tissue (activated by epinephrine and glucagon, inhibited by insulin) releases fatty acids and glycerol; lipoprotein lipase on capillary endothelium hydrolyzes triglycerides in circulating lipoproteins.

Phospholipids and sphingolipids

Glycerophospholipids have two fatty acids on glycerol carbons 1 and 2 and a phosphate on carbon 3 linked to a polar head group. They are the main membrane lipids.

• Phosphatidylcholine (lecithin): the most abundant membrane phospholipid, concentrated in the outer leaflet. Dipalmitoylphosphatidylcholine is the main component of lung surfactant; a lecithin to sphingomyelin ratio above about 2 in amniotic fluid indicates fetal lung maturity.
• Phosphatidylethanolamine and phosphatidylserine: inner leaflet. Phosphatidylserine flips outward in apoptosis, marking the cell for phagocytosis, and on activated platelets it supports clotting.
• Phosphatidylinositol: phosphorylated to PIP2, which phospholipase C cleaves into the second messengers IP3 and DAG.
• Cardiolipin: in the inner mitochondrial membrane; the antigen detected in nontreponemal syphilis tests.

Sphingolipids are built on sphingosine, an amino alcohol, instead of glycerol. Adding a fatty acid by an amide bond makes ceramide, the parent compound. Ceramide plus phosphocholine gives sphingomyelin, abundant in myelin and the only sphingolipid that is also a phospholipid. Ceramide plus one sugar gives a cerebroside (galactocerebroside in myelin, glucocerebroside elsewhere); ceramide plus an oligosaccharide with sialic acid gives a ganglioside, concentrated in neuronal membranes. Their lysosomal breakdown failures, the sphingolipidoses, are high yield.

Steroids and cholesterol

Steroids share a four fused ring nucleus (three six membered rings and one five membered ring). Cholesterol (27 carbons) has a hydroxyl at C3, which makes it weakly amphipathic, and a hydrocarbon tail. It is synthesized from acetyl CoA in the liver and other tissues, with HMG-CoA reductase as the rate limiting enzyme (inhibited by statins, suppressed by cholesterol, activated by insulin). Cholesterol is the precursor of three families: bile acids (the main route of elimination), steroid hormones (glucocorticoids, mineralocorticoids, androgens, estrogens, progestins) and vitamin D. In membranes, cholesterol slots between phospholipids and acts as a fluidity buffer: it stiffens membranes at high temperature and keeps them fluid at low temperature.

Eicosanoids

Eicosanoids are 20 carbon signaling lipids made from arachidonic acid, which phospholipase A2 releases from membrane phospholipids. Glucocorticoids inhibit this step (through lipocortin), blocking both branches below.

Aspirin irreversibly acetylates COX, so platelets, which lack a nucleus and cannot make new enzyme, lose thromboxane production for their lifespan of 7 to 10 days, while endothelial cells recover their prostacyclin synthesis. That imbalance is the basis of low dose aspirin's antiplatelet effect. Other NSAIDs inhibit COX reversibly. COX-1 is constitutive (gastric mucosal protection, platelet thromboxane, renal blood flow); COX-2 is inducible at sites of inflammation. Blocking COX can shunt arachidonic acid toward leukotrienes, explaining aspirin exacerbated respiratory disease.

Membranes and lipoproteins

The fluid mosaic model describes the membrane as a phospholipid bilayer with hydrophobic tails inward and polar heads facing water, studded with integral (transmembrane) and peripheral proteins and carbohydrate on the outer face (glycocalyx). Lipids and many proteins diffuse laterally; flipping between leaflets is slow and enzyme driven (flippases), so the two leaflets differ. Small nonpolar molecules (O2, CO2, steroids) cross freely, while ions and polar molecules need channels or carriers.

Because lipids do not dissolve in plasma, they travel in lipoproteins: a core of triglyceride and cholesteryl ester surrounded by a monolayer of phospholipid, free cholesterol and apolipoproteins. Larger particles carry more triglyceride and are less dense.

ApoC-II activates lipoprotein lipase, apoB-100 binds the LDL receptor, apoE mediates remnant uptake, and apoA-I activates LCAT, which esterifies cholesterol on HDL. In familial hypercholesterolemia (autosomal dominant, LDL receptor defects), LDL is very high, with tendon xanthomas (often on the Achilles tendon), corneal arcus and premature coronary disease.

Section 3Amino acids, peptides and proteins

Amino acids

Each of the 20 standard amino acids has a central α-carbon bonded to an amino group, a carboxyl group, a hydrogen and a distinctive side chain (R group). All except glycine (whose R group is hydrogen) are chiral, and proteins use the L form. At physiological pH, amino acids exist as zwitterions, with a protonated amino group (NH3+) and a deprotonated carboxyl (COO−). The isoelectric point (pI) is the pH at which the net charge is zero; for a simple amino acid it is the average of the two pKa values. Histidine, with an imidazole side chain pKa near 6, is the only residue that buffers well near physiological pH, which is why histidine rich hemoglobin is an important blood buffer.

The essential amino acids, which must come from the diet, are phenylalanine, valine, threonine, tryptophan, isoleucine, methionine, histidine, leucine and lysine (memory aid: "PVT TIM HaLL"); arginine is conditionally essential in growth. Leucine and lysine are purely ketogenic, several are both ketogenic and glucogenic (isoleucine, phenylalanine, tyrosine, tryptophan, threonine), and the rest are glucogenic. Tyrosine becomes essential in PKU.

The peptide bond

Amino acids join by a peptide bond, an amide bond formed by condensation between the carboxyl of one residue and the amino group of the next, releasing water. Chains are written from the free amino (N-terminus) to the free carboxyl (C-terminus), the same direction in which ribosomes build them. Resonance gives the C-N bond partial double bond character, so the bond is rigid and planar, usually in the trans configuration, and cannot rotate. Rotation is possible only around the bonds to the α-carbon (the φ and ψ angles), which constrains the shapes a chain can adopt. Peptide bonds are very stable and are broken by proteases or by strong acid with heat.

Levels of protein structure

• Primary: the amino acid sequence, held by covalent peptide bonds and dictated by the gene. It determines all higher levels. A single substitution, as in sickle cell disease, can change function.
• Secondary: local folding stabilized by hydrogen bonds between backbone C=O and N-H groups. The α-helix is right handed with 3.6 residues per turn and side chains pointing outward; proline and runs of glycine disrupt it. The β-pleated sheet links extended strands side by side, parallel or antiparallel. Turns and loops connect them.
• Tertiary: the complete three dimensional fold of one polypeptide, stabilized by side chain interactions: the hydrophobic effect (the main driving force, burying nonpolar residues), hydrogen bonds, ionic bonds (salt bridges), van der Waals forces and covalent disulfide bonds between cysteines, common in extracellular proteins such as antibodies and insulin.
• Quaternary: the arrangement of two or more polypeptide subunits, as in hemoglobin (four), collagen (three) and immunoglobulins (four chains). It allows cooperativity and allosteric control.

Proteins are broadly globular (compact, soluble, usually enzymes, transporters and antibodies) or fibrous (elongated, insoluble, structural, such as collagen, elastin and keratin). Folding is assisted by chaperones (heat shock proteins), and misfolded proteins are tagged with ubiquitin and degraded in proteasomes.

Collagen

Collagen is the most abundant protein in the body and the main tensile element of bone, tendon, ligament, skin, the annulus fibrosus and cartilage. Its sequence is a repeating Gly-X-Y triplet, where X is often proline and Y often hydroxyproline or hydroxylysine. Glycine at every third position is essential because only its tiny side chain fits in the crowded center of the triple helix, three left handed α chains wound into a right handed superhelix.

Synthesis spans the cell and the extracellular space:

1. Translation of pre-pro-α chains on the rough ER.
2. Hydroxylation of selected proline and lysine residues by prolyl and lysyl hydroxylase, which require vitamin C (ascorbate) and iron. Deficiency causes scurvy: weak collagen, bleeding gums, perifollicular hemorrhages, easy bruising and poor wound healing.
3. Glycosylation of hydroxylysine residues.
4. Assembly of three chains into a procollagen triple helix, stabilized by disulfide bonds at the C-terminal propeptides, then secretion.
5. Outside the cell, procollagen peptidases cleave the propeptides to give tropocollagen, which self assembles into fibrils.
6. Lysyl oxidase, a copper dependent enzyme, forms covalent cross links between lysine and hydroxylysine residues, giving tensile strength. Copper deficiency (Menkes disease) weakens collagen and elastin.

Memory aid: type I is in "bone" (b-ONE), type II in "car-TWO-lage", type III in "reticular" and type IV "under the floor" (basement membrane).

Elastin

Elastin lets tissues stretch and recoil: large arteries, lung, skin, the ligamentum flavum (whose yellow color comes from its high elastin content) and the ligamentum nuchae. It is rich in glycine, proline, alanine and valine, has little hydroxyproline and no hydroxylysine, and is not glycosylated. Tropoelastin is secreted and laid on a scaffold of fibrillin microfibrils, then cross linked by lysyl oxidase into desmosine cross links (four lysines), forming a rubber like network that recoils after stretch. Fibrillin defects cause Marfan syndrome (tall stature, long limbs, lens dislocation, aortic root dilation), and unopposed neutrophil elastase in α1-antitrypsin deficiency destroys alveolar elastin, causing panacinar emphysema.

Hemoglobin and myoglobin

Myoglobin is a single polypeptide with one heme group that stores oxygen in muscle. Hemoglobin (adult HbA, α2β2) is a tetramer with four hemes, each carrying one O2 on iron in the ferrous (Fe2+) state. Heme is a protoporphyrin ring; a proximal histidine binds iron from one side, and a distal histidine limits carbon monoxide binding. Both chains are mostly α-helix, a classic globular fold.

Myoglobin binds O2 with high affinity and a hyperbolic curve (P50 about 1 to 3 mmHg), so it releases oxygen only when muscle PO2 falls very low. Hemoglobin shows cooperativity: deoxyhemoglobin is in the low affinity T (tense) state, and binding of each O2 shifts the molecule toward the high affinity R (relaxed) state, producing a sigmoidal curve with a P50 of about 26 to 27 mmHg. The steep middle portion lets hemoglobin load fully in the lungs (PO2 near 100 mmHg) and unload a lot of oxygen in tissues (PO2 about 40 mmHg at rest, lower in working muscle).

A right shift (lower affinity, more unloading) is caused by increased CO2, H+ (the Bohr effect), temperature and 2,3-bisphosphoglycerate, all features of active muscle; 2,3-BPG also rises at altitude and in chronic anemia. A left shift is caused by the opposite changes and by fetal hemoglobin (α2γ2), which binds 2,3-BPG poorly and so takes oxygen from maternal blood. Carbon monoxide binds heme about 200 to 250 times more avidly than oxygen, lowers carrying capacity and shifts the remaining curve left. Methemoglobin has iron oxidized to Fe3+ and cannot carry O2. In sickle cell disease, a glutamate to valine substitution at position 6 of the β chain makes deoxygenated HbS polymerize.

Denaturation

Denaturation is loss of secondary, tertiary and quaternary structure with the primary sequence intact; peptide bonds are not broken. Causes include heat, extremes of pH, organic solvents, detergents, urea, heavy metals (which bind sulfhydryl groups) and mechanical agitation. Function is lost, solubility usually falls and the protein may aggregate (as egg white does when cooked). Some small proteins refold spontaneously when the denaturant is removed, which showed that sequence alone encodes the native fold. Misfolding with aggregation into β sheet rich fibrils underlies amyloidosis and the prion diseases.

Section 4Enzymes

How enzymes catalyze

Enzymes are biological catalysts, nearly all proteins (a few catalytic RNAs, or ribozymes, exist, including the peptidyl transferase of the ribosome). They speed reactions by factors of a million or more by lowering the activation energy, the energy barrier to the transition state. They do not change the free energy change (ΔG) of the reaction, the equilibrium constant or the final equilibrium position; they only help equilibrium arrive sooner, and they are not consumed. Whether a reaction is favorable is a matter of ΔG (negative means spontaneous), while how fast it goes depends on activation energy.

Substrate binds in the active site, a pocket formed by residues that may be far apart in the primary sequence. The induced fit model (an update of the lock and key model) holds that binding changes the shape of the enzyme to fit the transition state more tightly. Mechanisms include orienting substrates close together (proximity), straining bonds, acid base catalysis by side chains such as histidine, covalent catalysis (the serine of serine proteases) and metal ion catalysis. Enzyme activity depends on temperature (rising until denaturation, near 37 °C optimum in humans) and pH (pepsin works best near pH 2, trypsin near pH 8).

The six enzyme classes are oxidoreductases (dehydrogenases, oxidases), transferases (kinases, transaminases), hydrolases (proteases, lipases, phosphatases), lyases (add or remove groups without hydrolysis, such as aldolase and decarboxylases), isomerases and ligases (join molecules using ATP, such as DNA ligase and carboxylases). A kinase adds phosphate from ATP; a phosphatase removes it by hydrolysis; a phosphorylase adds inorganic phosphate to break a bond (glycogen phosphorylase).

Michaelis Menten kinetics

For a simple enzyme, E + S ⇌ ES → E + P, the initial reaction velocity follows the Michaelis Menten equation: v = Vmax[S] / (Km + [S]). Plotted against substrate concentration, velocity rises steeply and then plateaus in a hyperbola as the enzyme becomes saturated.

• Vmax is the maximum velocity at saturating substrate. It is proportional to enzyme concentration; adding more enzyme raises Vmax.
• Km (the Michaelis constant) is the substrate concentration at which v = ½ Vmax. It is a property of the enzyme and substrate and does not depend on enzyme amount. A low Km means high affinity: the enzyme is half saturated at low substrate.
• When [S] is much less than Km, velocity is roughly proportional to [S] (first order); when [S] is much greater, velocity approaches Vmax (zero order). Ethanol metabolism by alcohol dehydrogenase saturates at low blood levels, so it follows zero order kinetics.

A classic physiological comparison: hexokinase (most tissues) has a low Km for glucose and is inhibited by its product, glucose 6-phosphate, so tissues trap glucose even when blood sugar is low; glucokinase (liver and β cells) has a high Km and high capacity and is not inhibited by product, so the liver takes up glucose mainly after a meal and the β cell senses glucose across its physiological range.

The Lineweaver Burk plot

Taking the reciprocal of both sides gives a straight line: 1/v = (Km/Vmax)(1/[S]) + 1/Vmax. On this double reciprocal plot the y-intercept is 1/Vmax, the x-intercept is −1/Km, and the slope is Km/Vmax. The plot makes changes in Km and Vmax easy to read and is the standard way the board presents inhibitors. The farther right the x-intercept (closer to zero), the larger the Km and the lower the affinity; the higher the y-intercept, the lower the Vmax.

Types of inhibition

A memory device: competitive inhibitors compete for the active site, so flooding the system with substrate wins back full speed (Vmax unchanged) but it takes more substrate to get halfway there (Km up). Noncompetitive inhibitors effectively remove enzyme from service, so Vmax falls while the remaining enzyme behaves normally (Km unchanged).

Regulation: allosteric and covalent

Allosteric enzymes are usually multisubunit proteins with regulatory sites separate from the active site. Binding of an effector shifts the enzyme between low and high activity conformations, and cooperative substrate binding gives a sigmoidal velocity curve rather than a hyperbola. Positive effectors shift the curve left (activation) and negative effectors right. Allosteric enzymes typically catalyze the committed, rate limiting step of a pathway and are inhibited by the pathway's end product (feedback inhibition). The model example is phosphofructokinase-1 of glycolysis, inhibited by ATP and citrate (energy plentiful) and activated by AMP and fructose 2,6-bisphosphate (energy needed, insulin signal). Other rate limiting enzymes: HMG-CoA reductase (cholesterol), carnitine acyltransferase I (fatty acid oxidation, inhibited by malonyl CoA), acetyl CoA carboxylase (fatty acid synthesis), glycogen synthase and glycogen phosphorylase.

Covalent modification regulates activity more slowly but often more powerfully. Phosphorylation by kinases (for example protein kinase A, activated by cAMP after glucagon or epinephrine) and dephosphorylation by phosphatases (activated downstream of insulin) switch enzymes on or off: phosphorylation activates glycogen phosphorylase and inactivates glycogen synthase, so a single glucagon signal turns glycogen breakdown on and synthesis off. Proteolytic activation of zymogens is irreversible: trypsinogen, pepsinogen, chymotrypsinogen and the clotting factors are made inactive and switched on by cleavage where they are needed. Activity is also controlled by the amount of enzyme (induction and repression of genes) and by isozymes, different proteins catalyzing the same reaction with different kinetics or locations, such as the CK and LDH isozymes.

Cofactors and coenzymes

Many enzymes need a nonprotein helper. The protein alone is an apoenzyme; with its helper it is a holoenzyme. Inorganic cofactors are metal ions (Zn2+ in carbonic anhydrase and alcohol dehydrogenase, Mg2+ for nearly all ATP using kinases, Fe and Cu in cytochrome oxidase, Cu in lysyl oxidase, Se in glutathione peroxidase). Organic coenzymes are mostly derived from vitamins; a tightly bound coenzyme is a prosthetic group.

Clinical enzymes

Enzymes that normally work inside cells leak into plasma when cells are damaged, and the pattern of elevation points to the tissue.

• Creatine kinase (CK): CK-MM from skeletal muscle (rises after intense exercise, intramuscular injection, trauma, statin myopathy and rhabdomyolysis, and is very high in Duchenne dystrophy and polymyositis); CK-MB from heart; CK-BB from brain. Cardiac troponin, a regulatory protein rather than an enzyme, is now the preferred marker of myocardial injury.
• ALT is relatively specific to the liver; AST is also found in heart and muscle. An AST to ALT ratio above 2 suggests alcoholic liver disease.
• Alkaline phosphatase (ALP) comes mainly from bone (osteoblasts) and liver (bile ducts). Bone sources: growing children, healing fractures, Paget disease (very high), osteoblastic metastases (prostate), osteomalacia and hyperparathyroidism. Liver sources: cholestasis. A raised GGT alongside ALP indicates a liver source.
• Amylase and lipase rise in acute pancreatitis; lipase is more specific.
• LDH is nonspecific (hemolysis, tissue injury, lymphoma).
• Acid phosphatase (prostate) has been replaced by PSA, but it is historically tied to prostate cancer and osteoclast activity (tartrate resistant acid phosphatase).

Section 5Nucleotides and nucleic acids

Purines, pyrimidines and nucleotides

A nucleoside is a nitrogenous base joined to a pentose sugar by an N-glycosidic bond; a nucleotide is a nucleoside with one to three phosphates on the 5′ carbon. The purines, adenine (A) and guanine (G), have two fused rings (a six membered and a five membered ring). The pyrimidines, cytosine (C), thymine (T) and uracil (U), have a single six membered ring. Memory aids: "PURe As Gold" and "CUT the PYe". DNA contains A, G, C and T on 2′-deoxyribose; RNA contains A, G, C and U on ribose, whose 2′ hydroxyl makes RNA less stable and more reactive. Thymine is uracil with a methyl group; cytosine deaminates spontaneously to uracil, and having T rather than U in DNA lets repair enzymes recognize that damage.

Beyond nucleic acids, nucleotides are the energy currency (ATP, GTP), second messengers (cAMP, cGMP), parts of coenzymes (NAD+, FAD, coenzyme A) and activated carriers in synthesis (UDP-glucose for glycogen, CDP-choline for phospholipids, S-adenosylmethionine for methylation).

DNA and RNA structure

Nucleotides are linked by 3′,5′-phosphodiester bonds, giving each strand a sugar phosphate backbone with a negative charge and a direction from a free 5′ phosphate to a free 3′ hydroxyl. Sequences are written 5′ to 3′. The Watson Crick double helix (B-DNA) has two antiparallel strands wound in a right handed helix about 2 nm wide with about 10.5 base pairs per turn, a major groove and a minor groove where proteins read the sequence. Bases pair by hydrogen bonds inside the helix, A with T (two bonds) and G with C (three bonds); base stacking also stabilizes the helix. Chargaff's rules follow: in double stranded DNA, A = T, G = C, and purines equal pyrimidines. More GC content means a higher melting temperature, the point at which half the DNA has separated.

In eukaryotes, DNA wraps about 1.7 turns around a histone octamer (two each of H2A, H2B, H3 and H4) to form a nucleosome, with H1 binding linker DNA. Histones are rich in positively charged lysine and arginine. Loosely packed euchromatin is transcriptionally active; condensed heterochromatin is silent. Histone acetylation loosens chromatin and promotes transcription, while DNA methylation at CpG islands silences genes; both are epigenetic marks.

RNA is usually single stranded but folds into hairpins by internal base pairing. mRNA carries the code (least abundant, most heterogeneous); rRNA is the most abundant and forms the structure and catalytic center of ribosomes; tRNA is the smallest, with a cloverleaf shape, an anticodon loop and a 3′ CCA end where the amino acid attaches. Small nuclear RNAs function in splicing, and microRNAs silence genes.

Replication

DNA replication is semiconservative: each daughter duplex contains one parental and one new strand. It starts at origins (one in bacteria, many in eukaryotes) and proceeds in both directions from each origin. All DNA polymerases synthesize only 5′ to 3′, adding nucleotides to a free 3′ hydroxyl, and none can start a chain from nothing, so each new strand needs an RNA primer.

Because the two templates are antiparallel, one new strand, the leading strand, can be made continuously toward the fork, while the lagging strand is made in short Okazaki fragments away from the fork. Proofreading and mismatch repair together give an error rate of about one in a billion bases per replication.

Transcription

In transcription, RNA polymerase reads the template (antisense) strand 3′ to 5′ and builds RNA 5′ to 3′; the RNA matches the coding (sense) strand except that U replaces T. No primer is needed. In eukaryotes, RNA polymerase I makes rRNA, II makes mRNA (inhibited by α-amanitin from death cap mushrooms) and III makes tRNA and 5S rRNA; bacteria use a single polymerase (inhibited by rifampin). Polymerase II binds the promoter (including the TATA box about 25 bases upstream) with general transcription factors; enhancers and silencers, which can be far away, bind specific factors that raise or lower the rate.

The eukaryotic primary transcript (pre-mRNA) is processed in the nucleus: a 7-methylguanosine cap on the 5′ end, a poly-A tail on the 3′ end (after the AAUAAA signal), and splicing by the spliceosome (snRNPs) to remove noncoding introns and join coding exons. Alternative splicing lets one gene encode several proteins. Bacterial mRNA is not processed and is translated while still being transcribed.

Translation

The genetic code is read in nonoverlapping triplet codons, 5′ to 3′. Of 64 codons, 61 specify amino acids and 3 are stop codons (UAA, UAG, UGA). AUG codes for methionine and is the start codon (N-formylmethionine in bacteria). The code is degenerate (most amino acids have several codons, usually differing at the third, "wobble", position), unambiguous (each codon means only one amino acid) and nearly universal.

Each aminoacyl-tRNA synthetase attaches the correct amino acid to its tRNA using ATP; this charging step is where accuracy of the code is enforced. Eukaryotic ribosomes are 80S (60S + 40S); bacterial ribosomes are 70S (50S + 30S), the difference that many antibiotics exploit (aminoglycosides and tetracyclines on 30S; macrolides, chloramphenicol and clindamycin on 50S). During initiation, the small subunit and initiator tRNA find the start codon and the large subunit joins. In elongation, a charged tRNA enters the A site, the rRNA peptidyl transferase forms the peptide bond, and translocation (using GTP) moves the tRNAs to the P and E sites. At a stop codon, release factors free the polypeptide. The chain then folds and may be modified (phosphorylation, glycosylation, hydroxylation, cleavage).

Mutations and repair

Repair systems correct damage. Mismatch repair fixes replication errors (defective in Lynch syndrome, hereditary nonpolyposis colorectal cancer). Nucleotide excision repair removes bulky lesions such as UV induced thymine dimers (defective in xeroderma pigmentosum, with extreme sun sensitivity and skin cancers). Base excision repair removes single damaged bases such as deaminated cytosine. Double strand breaks are repaired by homologous recombination (BRCA1 and BRCA2) or error prone nonhomologous end joining.

Purine metabolism and uric acid

Purines are built de novo on a ribose 5-phosphate scaffold, activated as PRPP, from glycine, glutamine, aspartate, CO2 and two one carbon units from tetrahydrofolate, yielding IMP, which branches to AMP and GMP. Pyrimidines are made as a free ring (from carbamoyl phosphate and aspartate, via orotic acid) and then attached to PRPP. Ribonucleotide reductase converts ribonucleotides to deoxyribonucleotides (inhibited by hydroxyurea), and thymidylate synthase makes dTMP using folate (inhibited by 5-fluorouracil; methotrexate starves it by blocking dihydrofolate reductase). Because synthesis is costly, cells recycle free bases through salvage enzymes, chiefly HGPRT for hypoxanthine and guanine.

Purine degradation: AMP is converted to inosine (adenosine deaminase acts on adenosine), then to hypoxanthine; GMP gives guanine. Xanthine oxidase converts hypoxanthine to xanthine and xanthine to uric acid, the end product in humans, who lack uricase. Pyrimidines, by contrast, break down to soluble products (β-alanine, β-aminoisobutyrate, CO2 and ammonia) and do not form crystals. Adenosine deaminase deficiency causes a form of severe combined immunodeficiency, because toxic deoxyadenosine metabolites kill lymphocytes. Lesch-Nyhan syndrome (X-linked HGPRT deficiency) blocks salvage, so PRPP accumulates and drives purine overproduction, with hyperuricemia, gout, intellectual disability and self mutilation.

Hyperuricemia results from underexcretion (about 90 percent of cases: kidney disease, thiazide and loop diuretics, low dose aspirin, alcohol, lactic acidosis and ketosis competing for renal secretion) or overproduction (high cell turnover, tumor lysis, purine rich meats and seafood, fructose, enzyme defects). Above about 6.8 mg/dL urate exceeds its solubility, and monosodium urate crystals can deposit in cooler, distal joints and form tophi. Allopurinol and febuxostat inhibit xanthine oxidase; probenecid increases renal excretion; colchicine and NSAIDs treat the acute inflammation.

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