MCAT Biochemistry

1. Amino Acids, Peptides, Proteins

1.1. Proteinogenic Amino Acids
● AAs: amino, carboxy (CA) groups
○ In some AAs, amino and CA groups aren’t bonded to same C (e.g., GABA, with amino group on gamma C)
○ Proteinogenic AAs: 20 α-AAs encoded by codons in humans
○ Essential AAs: can’t be biosynthesized or can’t made by the body to meet demand. 9 in number.
■ “Very Heavy MILK WTF”: Val, His, Met, Iso, Leu, Lys, Trp, Thr, Phe
○ Nonessential AAs: can be biosynthesized or made by the body to meet demand.
■ “DEANS”: Asp, Glu, Ala, Asn, Ser
● Stereochemistry: all AAs are chiral, (S), ʟ-isomers
○ Except Gly, which is achiral b/c its side chain is H
○ Except Cys, which is (R) b/c ⦚–CH2SH > ⦚–COOH in priority order
● Hydrophobicity
○ Hydrophobic: nonpolar, hydrocarbon side chains (VALIF: Val, Ala, Leu, Iso, Phe)
■ Likely to be found in the interior of proteins, away from water on the surface of the protein
○ Hydrophilic: polar/charged AAs (Ser, Thr, Gln, Asn, Glu, Asp, Lys, Arg, His)
● Side-chain structure
○ Nonpolar, nonaromatic (GAVLIMP)
■ Glycine (Gly, G) ⦚–H
■ Alanine (Ala, A) ⦚–Me
■ Valine (Val, V) ⦚–iPr
■ Leucine (Leu, L) ⦚–CH2–iPr
■ Isoleucine (Iso, I) ⦚–CHMeEt
■ Methionine (Met, M) ⦚–CH2–CH2–SMe
● SMe and not SH, so nonpolar
● One of two aas with sulfur
■ Proline (Pro, P) ⦚–[pyrrolidine] (NH adj. to branch)
● Has a cyclic structure in the side chain
● Rigid ring, affects 2° structure i.e., makes kinks in alpha helices
○ Aromatic (FYW)
■ Tyrosine (Tyr, Y) ⦚–CH2–PhOH (para)
● Somewhat polar
● Hydroxyl is target for phosphorylation in post-transcriptional modification
■ Phenylalanine (Phe, F) ⦚–CH2Ph
 ■ Tryptophan (Trp, W) ⦚–CH2–[indole] (double bond of 5-membered ring adj. to branch)
● Precursor to serotonin, melatonin, and vitamin B3
○ Polar (STQNC)
■ Serine (Ser, S) ⦚–CH2OH
● Hydroxyl is highly polar, H-bonding
● Hydroxyl is target for phosphorylation in post-transcriptional modification
■ Threonine (Thr, T) ⦚–CH(Me)OH
● Hydroxyl is less polar than in Ser (due to Me), H-bonding
● Hydroxyl is target for phosphorylation in post-transcriptional modification
■ Cysteine (Cys, C) ⦚–CH2SH
● Can form disulfide linkages, with one of two aas with sulfur
● Has a thiol side chain which is less polar than hydroxyls in Ser/Thr (S is less EN than O)
● Thiol can be oxidized
■ Asparagine (Asn, N) ⦚–CH2–CONH2
● Also negatively charged
● Amide can’t be deprotonated
■ Glutamine (Gln, Q) ⦚–CH2–CH2–CONH2
● Also negatively charged
● Amide can’t be deprotonated
○ Acidic, (–) charged (DE)
■ Aspartate (Asp, D) ⦚–CH2–COO–
● CA is acidic
■ Glutamate (Glu, E) ⦚–CH2–CH2–COO–
● CA is less acidic than in Asp (extra alkyl is EDG)
○ Basic, (+) charged (HKR)
■ Lysine (Lys, K) ⦚–CH2–CH2–CH2–CH2NH3+
● Target for methylation and acetylation
■ Arginine (Arg, R) ⦚–CH2–CH2–CH2–NH–C(NH2)2+
● Protonating imine delocalizes (+) throughout all 3 N’s
■ Histidine (His, H) ⦚–CH2–[imidazole]+ (NH, double bond adj. to branch)
● Has a pKa near that of physiological pH
● Only imine is protonated at physiological pH (results in resonance)
1.2. Amino Acid Acid–Base
● Amphoteric: basic amino, acidic CA groups
○ Protonated (addition of a proton (H+)) in acidic soln. (low pH), deprotonated in basic soln. (high pH)
■ pH < pKa: most is protonated
■ pH = pKa: half of molecules are deprotonated, [HA] = [A–]
■ pH > pKa: most is deprotonated
 ○ (+)-charged in acidic soln.: ⦚–NH3+, ⦚–COOH
■ pKa of CA group ≈ 2
○ Zwitterion in neutral soln.: ⦚–NH3+, ⦚–COO–
■ (+)/(–), but electrically neutral
○ (–)-charged in basic soln.: ⦚–NH2, ⦚–COO–
■ pKa of amino group ≈ 9–10
● Titration
○ ⦚–COOH is more acidic than ⦚–NH3+
○ Add 0.5 equiv. base to acidic AA: buffer (pH = pKa, 1), flat curve
○ Add 1 equiv. base to acidic AA: all AAs are zwitterions, isoelectric point reached
■ pIacidic R = ½ (pKa, COOH + pKa, R) ≪ 6
■ pIneutral R = ½ (pKa, COOH + pKa, +NH3) ≈ 6
■ pIbasic R = ½ (pKa, R + pKa, +NH3) ≫ 6
○ Add 1.5 equiv. base to acidic AA: buffer (pH = pKa, 2), flat curve
○ NB = Isoelectric point of a peptide is the pH at which it net charge is 0
1.3. Peptide Bond Formation, Hydrolysis
● Peptides: multiple AAs/residues
○ Di/tripeptide: 2, 3 AAs
○ Oligopeptides: ≤ 20 AAs
○ Polypeptides: > 20 Aas
○ NB = These residues are joined together through peptide bonds (formed b/w -COO-group of AA and NH3+ of another AA
● Formation: ⦚–COO– + +H3N–⦚ ⇌ ⦚–CONH–⦚ + H2O
○ Condensation/dehydration, acyl substitution
■ Peptide bonds formation is a condensation/dehydration rxn that results in H20 removal
○ Partial double-bond character in C–N bond: delocalized π e– in carbonyl, amino N:
■ [⦚–(O=)C(–NH)–⦚] ↔ [⦚–(–O–)C(=N+H)–⦚]
■ Restricts rotation, rigid backbone
■ Other σ bonds are free to rotate
○ N-terminus (free amino end), C-terminus (free CA end)
■ Proteins are synthesized N to C
● Hydrolysis: reverse rxn. i.e., breaks peptide bond, consumes water
○ Hydrolytic enzymes are AA-/terminus-specific
● NB = 1. Net charge of a peptide/protein = sum of charges of its side chains and the charges of the N- and C-termini
2. At physiological pH, the N-terminus is + charged and the C-terminus is - charged, and the two charges cancel, leaving
only the side chain charge
3. Number of possible arrangements for peptide = n! where n is the number of AAs in the peptide
Examples: dipeptide 2! =2 (2x1); tripeptide 3! = 6 (3x2x1); tetrapeptide 4! = 24 (4x3x2x1)
1.4. Primary, Secondary Structure
● 1° structure: AA sequence, from N- to C-terminus
○ Covalent peptide bonds between adjacent AAs
○ Protein sequencing
● 2° structure: backbone interactions
○ H-bonding between backbone groups (carbonyl O, amide H); E.g., of 2° structure: α helix & β-pleated sheet
○ α helix: clockwise spiral
■ H-bonding 4 residues apart
■ Side chains point out from the helix
■ Pro: starts of α helices (rigid) or introduce a kink in the peptide chain when found int the middle of α helix
■ Gly: turns in α helices (conformationally flexible)
■ α helix is an important structure of keratin
○ β-pleated sheet: rippled sheet
■ Anti/parallel H-bonding peptide chains
■ Pro: turns in β-pleated sheets
■ Fibroin, primary protein component of silk fibers, is composed of β-pleated sheet.
1.5. Tertiary, Quaternary Structure
● Fibrous (sheets/long strands), globular (spherical) proteins
● 3° structure: Three-dimensional shape; side-chain interactions
○ Hydrophobic/philic interactions between side chains
■ Hydrophobic AAs inward, hydrophilic AAs outward
● Hydrophobic AAs outside: ΔH > 0 (break H bonds w/in water), ΔS < 0 (no solvation layer) → ΔG > 0
(nonspontaneous)
● Hydrophobic AAs inside: ΔS > 0 (solvation layer, more configurations for water) → ΔG < 0
(spontaneous)
○ Salt bridges: acid–base rxns., ionic bonds
○ Disulfide bonds: S–S, forms loops in peptide chain in oxidizing environment
■ Cys + Cys → cystine + 2 H+ + 2 e– (oxidation)
■ This bind can be broken by reducing agents such as β-mercaptoethanol or dithiothreitol
○ Molten globule: 3° structure formed
○ Denaturation: loss of 3° structure
○ Non-covalent interactions, such as hydrogen bonds, ionic bonding, and hydrophobic effects, allow proteins to fold into 2o
structures (α-helices and β-sheets) that combine to form tertiary structures.

● 4° structure: multiple subunits

○ Subunits: smaller globular proteins, e.g., hemoglobin and IgG antibodies
○ Allostery: conformational/structural change in 1 subunit can change other subunits’ activity
○ Reduce protein SA, reduce amt. DNA needed to encode protein, bring catalytic sites closer
 ○ Conjugated proteins: prosthetic groups (organic molecules, metal ions, etc.) covalently bonded to protein
■ Lipoproteins (lipids), glycoproteins (carbs), nucleoproteins (NAs)
○ NB = All proteins have elements of 1o, 2o, 3o; not all proteins have 4o structure
1.6. Denaturation
● Temperature ↑ = average KE ↑ can overcome hydrophobic interactions
● Solutes can break disulfide bridges (reduction), overcome H bonds
● Detergents can disrupt noncovalent bonds, solubilize proteins
● NB = Proteins lose functionality when their 3o structure unfolds (denaturation)

2. Enzymes
2.1. Biological Catalysis
● Catalysts
○ Change kinetics: Ea ↓, easier to form/more stable transition state (TS ‡), rxn. rate ↑
○ Thermodynamics of rxn. (ΔHrxn, Keq, ΔG) stay the same
■ However, Ea ↓ means optimal temp may be lower (less energy needed to reach Ea)
○ Not changed or consumed in rxn.
○ Sensitive to pH, temp
● Major classifications: “LIL HOT”
○ Ligases: responsible for joining or additions/syntheses between large, similar molecules
■ Require ATP
■ e.g., DNA ligase
○ Isomerases: rearrange bonds w/in molecule
■ Constitutional isomers, stereoisomers
■ e.g., phosphoglucose isomerase, TPI, aconitase, mutases
○ Lyases: cleavage of 1 molecule into 2 molecules
■ No hydrolysis (without H2O), no redox (without transfer of electrons)
■ e.g., synthases, decarboxylases, aldolases
 ● synthases catalyze the synthesis of 2 molecules into a single molecule
○ Hydrolases: catalyze the breaking of a compound into 2 molecules using addition of H2O (hydrolysis)
■ e.g., phosphatase, lipases, peptidases, nucleases
○ Oxidoreductases: catalyze oxidation-reduction (redox) rxn that involves the transfer of electrons
■ Has e–-carrier cofactors, like NAD+ or NADP+
■ Reductants (e– donors), oxidants (e– acceptors)
■ e.g., dehydrogenases, reductases, oxidases
● Dehydrogenation rxn involves the removal of hydrogen to form a double bond
○ Transferases: move functional groups between molecules
■ Kinases: transfer phosphate group (Pi) generally from ATP to another molecule
● NB = Enzymes lower the activation energy; enzymes do not alter the free energy (ΔG) or enthalpy (ΔH), rather they change the
rate (kinetics) at which equilibrium is reached.
2.2. Mechanisms
● Enzymes provide favorable microenvironments (pH, charge), stabilize TS‡, bring reactive groups closer together
● Enzyme–substrate binding
○ Enzymes posses an active site where catalysis occur and act on a molecule known as substrate
○ Substrate binds to active site, forms enzyme–substrate complex (ESC)
■ H bonding, ionic interactions, transient covalent bonds stabilize binding
○ Lock-and-key theory: substrate fits in active site w/ no conformational changes
○ Induced-fit model: upon binding, active site changes conformation to complement substrate
■ More accepted
● Cofactors/coenzymes: nonprotein molecules needed for effective activity
○ Cofactors (inorganic/metal): minerals, etc.
○ Coenzymes (organic): vitamins, vitamin derivatives
■ Water-soluble vitamins: easily excreted, must be replenished regularly
● B vitamins ("The RhiNo Paid Pill Boy For Coke")
○ B1: thiamine (TPP)
○ B2: riboflavin (FAD, FMN)
○ B3: niacin (NAD)
○ B5: pantothenate (CoA)
○ B6: pyridoxine
○ B7: biotin
○ B9: folate
○ B12: cobalamin
● C: ascorbate
■ Fat-soluble vitamins (ADEK): regulated by partition coefficients (non/polar solubilities)
● A: β-carotene, retinol/al/oic acid
● D: cholecalciferol, etc.
 ● E: tocopherols, tocotrienols
● K: phylloquinone, menaquinones
○ Apoenzymes (w/o cofactors), holoenzymes (w/ cofactors)
○ Prosthetic groups (tightly bound), cosubstrates (loosely bound)
2.3. Kinetics
● Enzyme activity = velocity = rate
● Saturation: enzyme at max velocity (vmax, mol enzyme/s), need more enzyme to speed up rxn.
○ i.e., as substrate concentration increases, the rxn rate increases until a maximum value is reached
○ Enzyme kinetics is dependent on: environmental condition, conc. of substrate and enzyme
● Michaelis–Menten (MM) kinetics
○ As the amount of substrate increases, the enzyme is able to increase its rate of reaction until it reaches a maximum
enzymatic reaction rate (vmax).
■ NB = Once vmax is reached, adding more substrate will not increase the rate of rxn.
○ MM equation: E + S ⥫(k1/k–1)⥬ ES ⤚(kcat)→ E + P
■ MM equation describes how the rate of the reaction, v, depends on the concentration of both the enzyme, [E], and
the substrate, [S], which forms product, [P].
○ Assumptions
■ In E + S ⇌ ES (⇌ EP →) E + P, rate-limiting step is ES ⇌ EP
■ [S] ≫ [E], [P]0 ≈ 0
■ d[E]/dt, d[S]/dt, d[ES]/dt are negligible
○ Michaelis constant (Km): enzyme affinity for substrate, intrinsic to enzyme/substrate
■ Km = [S] when v = ½ vmax (when 50% of enzyme is bound as ES)
● [S] < Km: changing [S] greatly changes v
● [S] > Km: changing [S] has little effect on v
■ Km = [E][S]/[ES] at steady state, Km = (kcat + k–1) / k1
● Different from binding constant: KD = [E][S]/[ES] at equilibrium, KD = k–1/k1
■ Comparing enzymes/substrates
● Low Km: high substrate affinity (less S needed for 50% saturation = faster v for given [S])
● High Km: low substrate affinity (more S needed for 50% saturation = slower v for given [S])
○ kcat: rate of ES → P i.e., the rate at which an enzyme bound substrate is converted to product
■ Low kcat (or low plateau): low turnover, slower rxn
■ High kcat (or high plateau: high turnover, faster rxn
■ NB = On the MM plot, catalytic turnover is reflected in the height of the plateau that occurs at high substrate conc.
■ Catalytic efficiency = kcat / Km is a measure if enzyme specificity
○ vmax = [E]0kcat
○ v = d[P]/dt = vmax[S] / (Km + [S])
■ v ≈ (kcat / Km) [E][S] when Km ≫ [S]
● MM plot: v vs. [S], hyperbolic
 ○ Plotting: (0, 0), (Km, ½ vmax), (∞, vmax)

● Lineweaver–Burk (LB) plot: 1/v vs. 1/[S], linear

○ Double reciprocal of MM plot
○ Plotting: intercept of line on x axis = –1/Km, and intercept of line on y axis = 1/vmax (–1/Km, 0), (0, 1/vmax), slope = Km /vmax
○ Used to calculate Km, vmax
○ NB = LB plot is useful when determining the type of inhibition that an enzyme is experiencing

● Cooperativity
○ v vs. [S] graph is sigmoidal
○ Cooperative enzymes have multiple subunits, multiple active sites
○ Low-affinity tense state (T), high-affinity relaxed state (R)
■ More substrate bound: (R) favored
■ Less substrate bound: (T) favored
○ Hill’s coefficient (n): measures cooperativity
■ n > 1: (+) cooperative binding, more ligands bind → enzyme affinity ↑
 ■ n = 1: no cooperativity
■ n < 1: (–) cooperative binding, more ligands bind → enzyme affinity ↓

2.4. Effects of Local Conditions on Activity

● Temp: usually, v doubles every +10 °C until optimal temp = 37 °C
○ Beyond optimal temp, v plummets: enzyme denatures
● pH: highest v at optimal pH = 7.4
○ Except in digestive tract: pepsin in stomach (optimal pH ≈ 2), pancreatic enzymes in duodenum (optimal
pH ≈ 8.5)
● Salinity: [salt] ↑ → v ↓
○ Salt disrupts H bonds/ionic interactions, partially changes enzyme conformation or denatures enzyme
2.5. Regulation
● Feedback regulation
○ (–) feedback: high [product] of pathway inhibits enzyme(s) upstream, avoid producing too much product
○ Feedforward regulation: preceding intermediates regulate enzyme
● Reversible inhibition
○ Competitive: inhibitor binds active site, competes w/ substrate
■ Km ↑ (more [S] needed for 50% saturation), no change in vmax (high enough [S] can outcompete inhibitor)
● Overcome by increasing [S] to outcompete inhibitor

■ LB plot: x-int. is smaller, lines intersect at y-axis

 ○ Noncompetitive: inhibitor binds allosteric site, changes enzyme conformation; binds with equal affinity to enzyme and ESC
■ No change in Km (affinity of uninhibited enzyme is the same), vmax ↓ (less enzyme available)
● Can’t overcome by increasing [S], since Km is unchanged
■ Inhibits E, ES equally

■ LB plot: y-int. is larger, lines intersect at x-axis

○ Mixed: noncompetitive, but diff. affinities for E, ES
■ Do not bind at the active site, but at an allosteric site
■ Km ↑ if inhibiting E is preferred, Km ↓ if inhibiting ES is preferred, vmax ↓
■ LB plot: lines intersect at point not on axes
○ Uncompetitive: inhibitor binds allosteric site of ES only
■ Km ↓ (removing ES from system drives Le Châtelier), vmax ↓ (less ES available)
■ LB plot: lines are parallel
● Irreversible inhibition
○ Inhibitor disables active site for a long time, or permanently
○ Overcome only by increasing [E]
● Regulated enzymes
○ Allosteric: activators/inhibitors bind allosteric sites
○ Covalently modified
■ Phosphorylation (covalent modification with phosphate): activate/deactivate enzyme
■ Glycosylation (covalent modification with carbohydrate): tag enzyme for transport, modify activity/selectivity
○ Zymogens: secreted in an inactive form and have regulatory domain must be cleaved/modified to activate enzyme
■ e.g., trypsinogen (inactive form) – trypsin (active form) involved in digestion, another e.g., caspases (apoptosis)

3. Nonenzymatic Protein Function

3.1. Cellular Functions
● Structural proteins: cytoskeleton/extracellular matrix (ECM), highly repetitive 2°/super-2° structure, and are fibrous in nature
○ Collagen: most of ECM. They provide strength, and flexibility
 ■ Most common protein in the body
■ Trihelical fibers characteristics: 3 left-handed helices woven into super-2° right-handed helix
● High Gly content (unusual for α helices), b/c structure packs AAs very close together
○ Elastin: stretch/recoil in skin, lungs, endothelia, etc.
■ Mostly A/G/V/P AAs
○ Keratin: intermediate filaments, cell mechanical integrity, regulation
■ Only in epithelia, most of hair and nails
■ α helices
○ Actin: microfilaments, thin filaments in sarcomeres, cell migration
■ Polarity: (+) away from, (–) toward nucleus
● Allows unidirectional movement along filament
■ Double helices
■ It is the most abundant protein in eukaryotic cells
○ Tubulin: microtubules, structure, segregate chromosomes in anaphase, intracellular transport, cell migration/motility
■ Polarity: (+) away from, (–) toward nucleus
■ 9+2 structure (9 doublet tubles, 2 central tubles) in flagella, 9 triplets in centrioles
● Motor proteins
○ Myosin: primary motor protein that interacts with actin and is involved in motor activity, and cellular transport
■ 1 head: neck movement = power stroke of sarcomere contraction
○ Kinesin: aligns chromosomes in metaphase, depolymerizes microtubules in anaphase
■ 2 heads: move in “steps,” 1 or both heads attached at all times
● ATP on attached head rotates kinesin → other head attaches when its ADP is removed →
original head detaches when its ATP is dephosphorylated
■ Vesicle transport: move toward (+) (away from nucleus), anterograde
○ Dynein: involved in cilia and flagella sliding movement (cell migration)
■ Vesicle transport: move toward (–) (toward nucleus), retrograde
● Binding proteins: bind and sequester/transport molecules
○ Sequester: high affinity for substrate
○ Transport: varying affinity depending on environment
○ e.g., hemoglobin, Ca2+-binding proteins, transcription factors
● Cell adhesion molecules (CAMs): are integral membrane proteins and helps cells to bind cell to ECM/other cells
○ Cadherins (Ca2+-dependent adhesion proteins): glycoproteins, mediate Ca2+-dependent cell adhesion
■ Hold similar cell types together
○ Integrins: bind cell to/communicate w/ ECM, cell signaling
■ Have two membrane-spanning α, and β chains
○ Selectins: allow cells to bind membrane carbohydrates, recruit cytokines/inflammation factors, allow WBC migration
■ On WBCs, blood vessel epithelia
● Antibodies/immunoglobulins (Ig’s)
○ 2 identical heavy chains, 2 identical light chains, held together by disulfide bonds and noncovalent interactions
 ○ Variable, diversity (in heavy chains), joining, constant regions
■ Antigen-binding region: V(D)J

○ Functions
■ Neutralization: block pathogen or toxin surface
■ Opsonization: mark pathogen for phagocytosis
■ Agglutination: clump antigens/antibodies into large insoluble protein complexes for phagocytosis
3.2. Biosignaling
● Ion channels: specific pathways for facilitated diffusion of ions
○ Ungated: unregulated and always open
■ e.g., K+ leak channels in all cells
○ Voltage-gated: regulated by Δ membrane potential near channel
■ e.g., V-gated Na+ channels in excitable cells, pacemaker channels in sinoatrial node
○ Ligand-gated: regulated by ligand-binding
■ e.g., GABAA receptors (ligand-gated Cl– channels) at postsynaptic membranes
● Enzyme-linked receptors: receptors that catalyze rxns. after binding ligand and usually initiates a 2 nd messenger cascade
○ Membrane-spanning domain: anchors receptor in membrane
○ Ligand-binding domain: binds ligand, induces conformational change, activates catalytic domain
○ Catalytic domain: often starts 2nd messenger cascade
● G protein-coupled receptors (GPCRs): signal transduction
○ Heterotrimeric G protein (a, b, g)
■ 7 membrane-spanning α helices
○ G-protein families
 ■ Gs: stimulates adenylyl cyclase, [cAMP] ↑
■ Gi: inhibits adenylyl cyclase, [cAMP] ↓
■ Gq: activates phospholipase C, cleaves PIP2 (phospholipid) from membrane, cleaves PIP2 into DAG + IP3
● IP3: opens Ca2+ channels in ER, [Ca2+] ↑
○ Trimeric G-protein cycle
■ Inactive G protein: α (w/ GDP), β/γ subunits
■ Ligand binds GPCR → receptor activated → α GDP is phosphorylated, activated α dissociates from β/γ
■ Activated α regulates adenylyl cyclase activity
■ Dephosphorylated α rebinds to β/γ, G protein inactivated
3.3. Protein Isolation
● Homogenize then centrifuge cell/tissue to isolate proteins by mass
● Electrophoresis
○ Put sample in electric field thru medium
○ Negatively charged compounds or anions migrate to positively charged anode, while positively charged compounds or
cations migrate to negatively charged cathode
○ For proteins and small molecules, the gel is polyacrylamide. For larger molecules (>500 bp), the gel is agarose
■ Migration velocity: v = Ez/f, where E = electric field, z = molecule net charge, f = frictional coefficient
■ Anode is (+), cathode is (–) b/c gel electrophoresis is an electrolytic cell (nonspontaneous)
○ Porous matrix medium
■ Small, highly charged molecules in large E = faster migration
■ Large, convoluted, neutral molecules in small E = slower migration
○ Native PAGE (polyacrylamide gel electrophoresis): proteins in native states (original structure) or non-denaturing
conditions
■ Can’t differentiate proteins w/ same mass:charge, mass:size ratios
■ Compares proteins of known similar mass by size, charge
● In other words, they are used to separate proteins based on charge and size
○ SDS-PAGE: PAGE method in denaturing condition where the detergent SDS (sodium dodecyl sulfate) is used
■ SDS disrupts all noncovalent interactions, binds proteins
● Denatures proteins, and erases original charge (i.e., gives them a uniform charge)
■ Separates proteins by size (i.e., molecular weight or mass only)
● Only E and f (∝ mass) affect v
○ Reducing SDS-PAGE: Exactly the same as SDS-PAGE, but with the addition of a reducing agent, b-mercaptoethanol,
which will reduce the disulfide bridges and result in a completely denatured protein
○ Isoelectric focusing: a gel electrophoresis method that separates protein on the basis of their relative contents of acidic
and basic residues (i.e., their isoelectric point)
■ basic at cathode, acidic at anode
■ When protein reaches region where pH = pI, protein becomes neutral, stops migrating
■ Separates proteins by pI only
 ■ Can run from either end
● Southern blotting: detection of a specific DNA sequence in a sample
● Northern blotting: detection of a specific RNA sequence in a sample
● Western blotting: detection of a specific protein in a sample
● Chromatography: separates protein mixtures on the basis of their affinity for a stationary phase or a mobile phase
○ Place sample on stationary phase/adsorbent (solid), run it w/ mobile phase/eluent (liquid)
○ Sample elutes, migrates at diff. speeds, partitions
■ High affinity for stationary phase: slow migration, high retention time
■ High affinity for mobile phase: fast migration, low retention time
○ Column chromatography: elute sample thru column filled w/ silica/alumina beads, using gravity. That is uses beads of
polar compound, like silica or alumina (stationary phase), with a nonpolar solvent (mobile phase)
■ the less polar the compound, the faster it can elute through the column (short retention time)
○ Ion-exchange chromatography: charged beads, retains oppositely charged proteins. For instance, a + charge column will
attract and hold a + charge protein as it passes through the column
■ Elute retained proteins w/ salt gradient
○ Size-exclusion chromatography: uses porous beads. Smaller proteins become trapped and elute slower, while large
proteins are not trapped and elute first
○ Affinity chromatography: beads coated with receptors or targets or antibodies, and retain specific proteins
■ Elute retained proteins w/ free receptors/targets/antibodies
3.4. Protein Analysis
● Structure
○ X-ray crystallography: isolate, crystallize protein, then measure e– densities thru X-ray diffraction pattern
■ In other words, the protein structure can be primarily determined through x-ray crystallography
○ Nuclear magnetic resonance (NMR) spectroscopy, can also be used
● Amino-acid sequence
○ Edman degradation: sequentially remove N-terminus AA, analyze w/ mass spec
■ Only for small proteins (≤ 70 AAs)
○ Proteases: cleave protein at specific AAs, analyze smaller fragments w/ electrophoresis or Edman degradation
■ For large proteins
■ Breaks disulfide links, salt bridges
● Activity
○ Monitor known rxn. w/ given [S], compare rate to a standard
● Concentration
○ Protein concentration is determined calorimetrically, either by UV spectroscopy, or through color change rxns (such as
bicinchoninic acid (BCA) assay, Lowry reagent assay, and Bradford protein assay)
■ Bradford protein assay
● Mix protein w/ Coomassie Brilliant Blue dye (protonated, green-brown)
● Dye deprotonates on binding, turns blue
 ● [blue dye] ↑ = [protein] ↑
● Most used, but valid only for samples w/ 1 protein

4. Carbohydrates
4.1. Classification
● General formula: Cn(H2O)m
● Monosaccharides
○ Trioses (3 carbons), tetroses (4 carbons), pentoses (5 carbons), hexoses (6 carbons), etc.
○ Aldoses: have aldehyde as their most oxidized group
■ Simplest aldose: glyceraldehyde, CHO–(H)C(OH)–CH2OH
● The carbonyl carbon is the most oxidized and has the lowest possible number
● In an aldose, the aldehyde carbon will always be carbon number one (C-1)

○ Ketoses: have ketone as their most oxidized group

■ Simplest ketose: dihydroxyacetone, CH2OH–CO–CH2OH
● The carbonyl carbon is the most oxidized; the lowest number it can be assigned is carbon number 2 (C-2)
○ Ketoses can also participate in glycosidic bonds at this carbon C-2

○ Common sugars: fructose, glucose, galactose, mannose

● Stereochemistry
○ Optical/stereoisomers: same connectivities, diff. spatial arrangement of atoms
■ Enantiomers: mirror images but nonsuperimposable
● Chiral C: C w/ 4 diff. groups
 ● # stereoisomers = 2(# chiral C’s in molecule) that is 2n

■ Diastereomers: are not mirror images

● Epimers: are different at 1 chiral center only. E.g., d-ribose and d-arabinose, which only differ at C-2

○ Absolute configuration
■ (R)/(S): CIP priority rules, (R) is Clockwise, (S) is Counterclockwise
■ (+)/(–): experimentally determined; (+) = d, (-) = l
■ ᴅ/ʟ: hydroxyl of penultimate C in Fischer projection (highest-number chiral C), ᴅ is right, ʟ is left
● In other words, sugars with the highest-numbered chiral carbon with the –OH group on the right, are d-
sugars, while those with the –OH on the left are l-sugars
○ Fischer projection
■ Horizontal lines are wedges (out from page), vertical lines are dashes (into page)

■ Highest-oxidized group is nearest to the top, start counting from that topmost C (C-1)
4.2. Cyclic Sugars
● Cyclization: describes the ring formation of carbohydrates from their strain chain forms
● Intramolecular ring formation: hydroxyl O (Nuc) attacks carbonyl C
○ Forms cyclic hemiacetals (from aldoses), hemiketals (ketoses)
■ Furanoses (5-membered), pyranoses (6-membered)
○ Carbonyl carbon (becomes anomeric carbon) becomes chiral
 ■ Anomers: epimers at anomeric C
● α: anomeric C ⦚–OH is trans (axial) to ⦚–MeOH
○ In other words, the -OH on the anomeric carbon is trans to the free-CH2OH group

● β: anomeric C ⦚–OH is cis (equatorial) to ⦚–MeOH (“what’s up, bitch”)

○ In other words, the -OH on the anomeric carbon is cis to the free-CH2OH group

● Haworth projection: represent 3D structure of monosaccharide

○ Cyclic sugars are planar rings, w/ groups above/below the plane
○ Left/right in Fischer = up/down in Haworth
○ ᴅ/ʟ: abs. configuration highest-number chiral C, ᴅ is (R), ʟ is (S)
● Mutarotation
○ Exposure to water (cat. acid/base) → spontaneous ring opening/closing → mutarotation between α, β
anomer
■ Mutarotation involves the interconversion between α, β anomer via ring opening and reclosing
○ In soln., β-glucose is more stable: equatorial ⦚–OH avoids 1,3-diaxial interactions
○ As solid, α-glucose is more stable: anomeric effect between O lone pairs, EN ⦚–OH (?)
■ Not very well understood, beyond scope of MCAT?
4.3. Monosaccharides
● Single carbohydrate units. Can undergo redox rns, esterification, and glycoside formation
● Redox
○ Reducing sugars: hemiacetal cyclic sugars
■ Aldonic acid: open-chain aldose after oxidation (aldehyde → CA), i.e., aldoses can be oxidized to
aldonic acids
■ Lactone: cyclic aldose after oxidation (hemiacetal → ester) (anomeric C hydroxyl → carbonyl)
■ Alditol: aldose after reduction (aldehyde → alcohol), i.e., aldoses can be reduced to alditols
■ Deoxy sugar: sugar w/ ⦚–H instead of ⦚–OH
 ○ Indicators for reducing sugars (aldoses, enol tautomer of ketoses)
■ Tollens’ reagent: diamminesilver (I)
● Preparation
○ 2 AgNO3 (aq) + 2 NaOH (aq) → Ag2O (s) + 2 NaNO3 (aq) + H2O (l)
○ Ag2O (s) + 2 NH3 (aq) + NaNO3 (aq) + H2O (l) → 2 [Ag(NH3)2]+ NO3– (aq) + 2 NaOH (aq)
● Creates Ag “silver mirror” in presence of reducing sugars
■ Benedict’s reagent: Cu(OH)2, etc.
● Precipitates red Cu2O in presence of reducing sugars
■ Glucose oxidase: tests for glucose only
● Esterification
○ Hydroxyls + CAs/CA derivs. → esters
■ In other words, sugars react with carboxylic acids, and their derivatives to form esters

○ Phosphorylation: is a similar rxn in which a phosphate ester is formed by transferring a phosphate group from ATP onto a
sugar

● Glycoside formation:
○ The basis for building complex carbohydrates and requires the anomeric carbon to link to another sugar
■ Example: Hemiacetals or hemiketals + alcohols + cat. acid → acetals or ketals (glycosides)
● Furanosides: are glycosides derived from furanoses
● Pyranosides: are glycosides derived from pyranoses
○ Glycosidic bonds: [sugar]–OR
■ Glycoside formation is a dehydration reaction; thus, breaking a glycosidic bond requires hydrolysis
■ Disaccharides and polysaccharides form as a result of glycosidic bonds between monosaccharides
4.4. Complex Carbohydrates
● Disaccharide: 2 monosaccharides, joined by glycosidic bond
○ Anomeric C ⦚–OH can react w/ any ⦚–OH on another sugar
○ Sucrose: Glc-α-1,2-β-Fru

○ Lactose: Gal-β-1,4-Glc

○ Maltose: Glc-α-1,4-Glc

● Polysaccharide: monosaccharide chains, joined by glycosidic bonds

○ Homopolysaccharide: made of 1 monosaccharide
○ Heteropolysaccharide: made of multiple monosaccharides
○ Cellulose: β-ᴅ-glucose, β-1,4 bonds
■ Cellulase: needed to hydrolyze cellulose, missing in humans but found in certain animals like cows, and goats
● Humans cannot digest any β-linked sugars
○ Starch: α-ᴅ-glucose; function as main storage form for plants
■ Amylose: α-1,4 bonds only, linear and unbranched
■ Amylopectin: α-1,4 and α-1,6 bonds, branched
■ Amylase: hydrolyzes starch
● α-amylase: cleaves any α-1,4 bond randomly
○ Yields small sugars
● β-amylase: cleaves every 2nd α-1,4 bond from nonreducing end (w/o available anomeric C)
○ Yields maltose only
● γ: cleaves α-1,4 bond from nonreducing end
○ Yields glucose only
■ Iodine is indicator
 ○ Glycogen: α-ᴅ-glucose, has more α-1,6 bonds than starch; function as main storage form for animals
■ Highly branched = energy efficiency ↑, solubility ↑, breakdown from many sites
■ Glycogen phosphorylase: cleaves glucose-1-P from glycogen
● Hers disease: glycogen phosphorylase deficiency → no glycogenolysis → hepatomegaly,
hypoglycemia

5. Lipids
5.1. Structural Lipids
● Phospholipids
○ Phosphate, alcohol (hydrophilic), fatty acid (FA) tails (hydrophobic)
○ Saturation
■ Saturated FAs: C–C single bonds only
● Stronger van der Waals forces, solid at room temp, less fluid
■ Unsaturated FAs: C=C double bonds
● Harder to pack, liquid at room temp, more fluid
● Glycerophospholipids/phosphoglycerides: glycerol (propanetriol) backbone
○ 2 FA tails linked by ester bonds
○ 1 head group linked by phosphodiester bond
■ Phosphatidylcholine: choline head group (⦚–O–CH 2–CH2–NMe3+)
■ Phosphatidylethanolamine: ethanolamine head group (⦚–O–CH 2–CH2–NH2)
● Sphingolipids: sphingosine/oid backbone
○ 1 FA tail linked by amide bond
■ Backbone itself is also a long-chain FA
○ 1 head group at 1-hydroxyl
■ Ceramide: H head group
■ Sphingomyelins: phosphatidylcholine/ethanolamine head group, linked by phosphodiester bond
● Sphingophospholipids
● Neutral, make up plasma membranes of myelinating cells
■ Glycosphingolipids: sugar head group, linked by glycosidic bond
● Not a phospholipid
● Neutral, found on outer surface of cell membranes
● Cerebrosides: 1 sugar as head group
● Globosides: multiple sugars as head group
■ Gangliosides: oligosaccharide head group, w/ sialic acid(s) (N-acetylneuraminic acid, NANA) at terminus
● Glycolipids, so not a phospholipid
 ● (–) charge, used for cell communication/recognition, signal transduction
● Waxes: esters of long-chain FAs w/ long-chain alcohols
○ Malleable solids at room temp
○ Repel water, lubricate, prevent dehydration
5.2. Signaling Lipids
● Terpenes, terpenoids: precursors for signaling lipids
○ Terpenes: built from isoprene (2-methyl-1,3-butadiene, H2C=C(Me)–CH=CH2)
■ Monoterpenes: 2 isoprenes
■ Sesquiterpenes: 3 isoprenes
■ Diterpenes: 4 isoprenes (e.g., retinol)
■ Triterpenes: 6 isoprenes
■ Tetraterpenes: 8 isoprenes (e.g., carotenoids)
■ Polyterpenes: many isoprenes (e.g., natural rubber)
○ Terpenoids: terpene derivs.
● Steroids: 3 cyclohexanes, 1 cyclopentane fused together
○ Steroid hormones: bound to carrier proteins, secreted into bloodstream, pass thru cell membrane (and nucleus)
○ Cholesterols
■ Found in plasma membranes, allow membrane fluidity
● Prevent solidifying in cold temps/hyperpermeability in high temps
■ Precursors for steroid hormones, bile acids, vit. D
● Prostaglandins: arachidonic acid derivs., unsaturated CAs w/ 1 cyclopentane
○ Para/autocrine signaling, regulate cAMP synthesis
○ Downstream effects: smooth muscle function, circadian rhythm, fever
○ Nonsteroidal anti-inflammatory drugs (NSAIDs): inhibit cyclooxygenase (COX) → prostaglandins ↓
● Fat-soluble vitamins
○ Vitamin A: carotene, retinal (aldehyde, light-sensing), retinol (alcohol, storage), retinoic acid (CA, hormone)
○ Vitamin D: cholecalciferol
■ Calcitriol: vit. D activated in liver/kidneys, intestinal Ca2+/PO43– uptake ↑ → osteogenesis
■ Rickets (vit. D deficiency): underdeveloped long bones, stunted growth
○ Vitamin E: tocopherols, tocotrienols (aromatic ring w/ long isoprenoid side chain)
■ Antioxidants: aromatic ring reacts w/ free radicals, prevents oxidative damage
○ Vitamin K: phylloquinone (vit. K1), menaquinones (vit. K2)
■ Post-translational modifications to form prothrombin (cleaved to form clotting factor thrombin)
● Warfarin: blood thinner, vit. K recycling ↓
■ Introduce Ca2+-binding sites
■ “Vit. K is for koagulation, kalcium”
5.3. Energy Storage
● Fat: stores energy better than sugar
○ High energy density: more reduced C’s → more oxidation possible
■ ~9 kcal/g, compared to ~4 kcal/g for carbs/proteins/ketone bodies
○ Hydrophobic: no hydration needed for stability → weight ↓
○ Insulation: retains body heat
○ But slower to recruit than glycogen
● Triglycerides/triacylglycerols: 3 FAs linked to glycerol backbone by ester bonds
○ Depots of metabolic fuel in cytosol
○ Adipocytes: fat storage under skin, in breast, abdominal cavity
● Saponification: hydrolyze esters in triglycerides
○ Triglyceride + 3 lye (NaOH) → glycerol + soap (free FA salts)
■ Free FAs: unesterified FAs w/ carboxylate groups, bind to serum albumin
○ Surfactant: surface tension ↓, dissolves both hydrophilic/phobic molecules
■ Water + fats + soap → colloid, amphipathic soaps form micelles around fats

6. DNA
6.1. Structure
● Nitrogenous bases
○ Purines (2 rings): adenine (A), guanine (G)
○ Pyrimidines (1 ring): cytosine (C), thymine (T), uracil (U)
■ “CUT the pye,” and pies have 1 ring
● Nucleoside: pentose and nitrogenous base, linked by glycosidic bond (C-1′ sugar to N-9 purine/N-1 pyrimidine)
● Nucleotide: nucleoside and 1–3 phosphates, linked by phosphodiester bonds (C-5′ sugar)
○ Pyrophosphate/phosphoanhydride bonds: phosphate–phosphate
■ High energy: electrostatic repulsion between (–)-charged phosphates
■ Breaking bonds is always endothermic, but cleaving Pi is exothermic
● Hydrolysis: ΔHbreaking (P)–(P) bond + ΔHforming (P)–water bond < 0
○ Deoxyribose: ribose w/ no ⦚–OH at C-2
○ Dideoxyribose: ribose w/ no ⦚–OH at C-2, C-3
● Nucleic acids: nucleotide polymers, linked by phosphodiester bonds
○ Ribonucleic acid (ssRNA), deoxyribonucleic acid (dsDNA)
○ Sugar–phosphate backbone: phosphate links C-3′ to C-5′
■ Nucleic acids are synthesized 5′ to 3′
○ (–) charge due to phosphates
● DNA double helix (Watson–Crick model)
○ Antiparallel ssDNA strands, backbone facing outward
○ Complementary base pairing: A–T (2 H bonds), G–C (3 H bonds)
○ Chargaff’s rules: # purines = # pyrimidines
■ Formed before base-pairing was known, doesn’t say %A = %T, %G = %C
○ B-DNA: right-handed helix, turns every 3.4 nm (~10 bases)
■ Major (protein binding sites), minor grooves
■ Most DNA
○ Z-DNA: left-handed helix, turns every 4.6 nm (~12 bases)
■ Uncommon/unstable, found in GC-rich DNA or high salinity
● Denaturation: heat, basic pH, formaldehyde, urea disrupt H bonds, separate ssDNA strands
● Reannealing: spontaneous complementary base pairing
6.2. Eukaryotic Chromosome Organization
● Human DNA: ~6 billion bp, 46 chromosomes
● Chromatin: “beads on a string”
○ DNA wrapped around histones (nucleoproteins)
■ Histone core: H2A, H2B, H3, H4
■ H1: keeps wrapped DNA in place, stabilizes chromatin
■ High Lys, Arg content: (+) charge attracts (–)-charged DNA backbone
○ Nucleosome: ~200 bp wrapped around histone core
○ Heterochromatin: dense, dark in light microscopy, silent
■ Highly repetitive sequences
○ Euchromatin: uncondensed, light in light microscopy, expressed
● Telomeres: TTAGGG repeats at ends of DNA
○ DNA replication can’t reach ends: DNA Pol falls off for lagging strand
○ Shortened w/ each replication → cellular aging
■ Hayflick limit: # times a cell can replicate before telomeres are too short for cell division
■ Telomerase: extends telomeres, not produced by most somatic cells
○ High GC-content: strong strand attractions at ends of DNA, prevents unraveling
● Centromeres: heterochromatin region at center of DNA
○ GC-rich tandem repeats
○ Site where 2 sister chromatids connect
6.3. Replication
● Initiation
○ Replisome/replication complexes assemble at each origin of replication (ori)
■ Prokaryotes
● Circular DNA, small genome → 1 ori, 2 replication forks
 ■ Eukaryotes
● Linear DNA, large genome → many ori’s, many replication forks to speed up replication
● Sister chromatids remain connected at centromere
● Elongation: reads 3′ to 5′, synthesizes daughter strands 5′ to 3′, semiconservative (1 parent strand in each daughter dsDNA)
○ Helicase: disrupts base–base H bonds, separates dsDNA into ssDNA strands
■ ss-binding proteins: prevent ssDNA from reannealing, prevent unwanted interactions, protect ssDNA from
nucleases
● SSB (prok.), rep. protein A (RPA, euk.)
■ Topoisomerases: relax supercoils upstream (ahead of helicase), prevent torsional pressure
● Induce ss/ds breaks, re-ligate DNA to prevent overwinding double helix
● Gyrase (prok.): targeted by broad-spectrum fluoroquinolone (…-floxacin) antibiotics
○ Priming: DNA-dependent RNA Pol, forms initial RNA primer to polymerize from
■ DNA cannot be synthesized de novo
■ Leading strand is primed once, lagging strand is primed every Okazaki fragment
■ Primase (prok.), DNA Pol α (euk., also adds some ssDNA after RNA primer)
○ Synthesis: 5′ to 3′
■ Strand’s 3′ ⦚–OH attacks free nucleotide’s 5′ phosphate
● Releases PPi → 2 Pi, drives Le Châtelier
■ Sliding clamp: strengthens DNA Pol–template strand interactions
● DNA Pol III β dimer (prok.), PCNA trimer (euk.)
● Loaded by clamp loader: DNA Pol III subunits (prok.), rep. factor C (RFC, euk.)
■ Leading, lagging strands
● Okazaki fragments: 1,000–2,000 bp (prok.), 100–200 bp (euk.)
● DNA Pol III (prok.), DNA Pol δ/ε (euk.), DNA Pol γ (mitochondria)
○ Removing primers, ligating nicks
■ 5′-to-3′ exonucleases: DNA Pol I (prok.), RNase H (euk.)
■ Fill in missing dNTPs: DNA Pol I (prok.), DNA Pol δ (euk.)
■ Ligate nicks/fragments: DNA ligase
○ Proofreading during replication
■ 3′-to-5′ exonucleases: all DNA Pol’s (prok.), DNA Pol δ/ε (euk.)
● Termination
○ Prokaryotes
■ Tus–Ter: Tus protein binds Ter sites, ensures one-way replication forks
■ Termination occurs at desired site
○ Eukaryotes
■ Replication terminates when replication forks meet
■ Telomeres cannot be fully replicated on lagging strand
● Okazaki fragments need primers upstream to elongate
● Okazaki fragments need DNA upstream to remove primer, else RNA primer is degraded
6.4. Repair
● Cancer: excessive division w/o stimulus or controls, migrates locally or systemically (metastasis)
○ Oncogenes: mutated genes that cause cancer
■ Proto-oncogenes: usually cell-cycle genes
■ 1 mutated allele is enough for tumor growth
○ Tumor suppressors/anti-oncogenes: stop tumor progression
■ p53, Rb, etc.
■ 2 mutated alleles needed for cancer
● Proofreading: fixes mismatches during replication (S phase)
○ Incorrect complementary base-pairing → unstable H bonds
○ 3′-to-5′ exonuclease: excises incorrect base on daughter strand (unmethylated)
○ Mutations are more common on lagging strand
■ DNA ligase lacks proofreading
■ Many more RNA primers, which are more error-prone
● Mismatch repair (MMR): fixes mismatches in G2
○ MutS, MutL (prok.), MSH2, MLH1 (euk.)
● Nucleotide excision repair (NER): fixes UV-induced damage in G1/G2
○ UV → cyclobutane pyrimidine dimer (CPD) lesion → bulge in DNA strand
○ Excision endonuclease: nicks backbone on both sides of CPD, removes oligonucleotide
■ Uvr’s (prok.), TFIIH, XPG, etc. (euk.)
○ Fill in 5′ to 3′ using undamaged strand as template (no primer needed), ligate nick
● Base excision repair (BER): fixes small, non-helix-distorting lesions in G1/G2
○ Heat → cytosine deaminates, turns into uracil
○ Glycosylase recognizes, removes base
■ Apurinic/apyrimidinic (AP)/abasic site: backbone w/o base
○ AP endonuclease excises neighboring sequence
○ Fill in 5′ to 3′, ligate nick
6.5. Biotechnology
● Restriction enzymes/endonucleases: recognize, cut specific dsDNA sequences
○ Many are palindromic: 5′ to 3′ = antiparallel 3′ to 5′
○ Sticky ends: overhangs of ssDNA
○ Blunt ends: no overhang
○ Types
■ I: randomly cuts far away (> 1,000 bp) between 2 recognition sites
● Needs ATP (to move along DNA), Mg2+, S-adenosyl Met (SAM)
■ II: specifically cuts close to or w/in recognition site (most common)
● Needs Mg2+, usually palindromic
■ III: randomly cuts between 2 inverse recognition sites (rare)
 ● Needs ATP (to move along DNA), Mg2+, SAM
■ IV: cuts methylated DNA
■ V: cuts using guide RNA (e.g., Cas9)
● DNA cloning: amplifies small sample sequence w/ a vector (bacterial, viral plasmid)
○ Cut vector w/ restriction enzyme
■ Typically at multiple cloning site (MCS), which has many known unique restriction sites
○ Insert foreign DNA, ligate
○ Transform competent bacteria w/ vector, grow into colonies
○ Isolate transformed colonies
■ Screening: visual indicator of transformation
● Blue–white screen: recombination disrupts lacZ → no β-galactosidase expression → no X-gal
hydrolysis
○ White colonies: recombinant vector (can’t hydrolyze X-gal into blue product)
○ Blue colonies: nonrecombinant vector, off-target recombination or no transformation
■ Selection: kill non-transformed bacteria
● Antibiotic-resistance gene in plasmid
○ Produce lots of recombinant protein, or lyse cells and use restriction enzyme to isolate lots of cloned DNA
● DNA library: large collection of known DNA sequences
○ Genomic library: coding, noncoding regions
■ Chromosomal DNA, cut w/ restriction enzymes
○ Expression library: coding regions only
■ Complementary DNA (cDNA): reverse-transcribed processed mRNA
■ Complete, expressible gene sequences
● Polymerase chain reaction (PCR): amplifies DNA sequence
○ Melt dsDNA into single strands w/ heat
○ Anneal complementary primers, which hybridize
■ GC-rich, lower temp for more stability
○ Extend w/ free dNTPs and Taq Pol
■ Taq Pol: thermostable DNA Pol I, from thermophilic Thermus aquaticus bacteria
● Gel electrophoresis: separates DNA by sequence length
○ All DNA is (–)-charged b/c of backbone phosphates, migrate to anode
■ In all cells, anode attracts anions
■ In electrochemical cells, anode is (+)
○ Longer DNA migrates slower thru agarose gel
○ Plasmid thru gel electrophoresis
■ Supercoiled: travels faster than circular plasmid (much smaller size)
■ Linear: travels a little faster than circular plasmid (less bulky), migrates distance expected from DNA length
● Southern blot: detects, quantifies DNA strands
○ Cut DNA w/ restriction enzymes, separate fragments w/ gel electrophoresis
 ○ Transfer fragments to nitrocellulose membrane w/ pressure (capillary action), bake fragments onto membrane
○ Probe membrane w/ labeled ssDNA hybridization probes
○ Wash excess probe, visualize probed DNA
● Sanger sequencing
○ Replicate DNA w/ small amounts of fluorescent dideoxyribonucleotides (ddNTPs)
■ No ⦚–OH at C-2′ or C-3′, elongation cannot occur
○ Separate resulting fragments w/ gel electrophoresis
○ Read each base in order
● DNA technology applications
○ Gene therapy: transduces functional gene into human cells w/ modified retroviruses
■ Vector can infect cells but not replicate
■ Randomly integrated DNA can disrupt proto-oncogenes/tumor suppressors, chance of cancer ↑
○ Transgenesis: microinjects cloned gene (transgene) into fertilized ova/embryonic stem cells
■ Alters germ line, produces transgenic offspring
■ Knockout: delete/disable a specific gene
■ Chimera: inject transgenic embryonic stem cells into developing blastocysts

7. RNA
7.1. Genetic Code
● Central dogma: (replication) ⟳ DNA ⤚(transcription)→ RNA ⤚(translation)→ protein
● RNA types
○ Ribosomal RNA (rRNA): forms peptide bonds in ribosome, splices introns
■ Transcribed by RNA Pol I
■ Ribozymes: enzymatic RNA
○ Messenger RNA (mRNA): info for AA sequence
■ Transcribed by RNA Pol II from DNA template strand
● mRNA sequence is identical to coding strand (except T → U)
■ Polycistronic (prok.): 1 mRNA can encode multiple proteins
■ Monocistronic (euk.): 1 mRNA → 1 protein
○ Transfer RNA (tRNA): delivers corresponding AA to ribosome
■ Transcribed by RNA Pol III
■ Anticodon: complementary to codon
■ Aminoacyl-tRNA (aa-tRNA) synthetase: charges/activates tRNA at 3′ ⦚–OH w/ correct AA
● Charging requires ATP → AMP + PPi
● Codon: 3 bases = 1 AA
 ○ 61 codons encode AAs, 3 encode stop codons
○ Start codon (AUG): Met, starts translation
○ Stop codons (UAA, UGA, UAG): bind release factors instead of tRNA, stops translation
■ “U Are Annoying, U Go Away, U Are Gone”
○ Degeneracy: many codons encode 1 AA
■ Wobble position: 3rd base is variable
● Mutations are silent/degenerate/synonymous
● Redundancy → more fault tolerance for point mutations
○ Anticodon on tRNA: pairs w/ complementary, antiparallel codon on DNA
● Point mutations
○ Silent: mutated codon encodes same AA (degenerate)
○ Missense: mutated codon encodes different AA
○ Nonsense/truncation: mutated codon encodes stop
○ Frameshift: base is inserted/deleted (indel), entire reading frame shifts
7.2. Transcription
● DNA must be transcribed before leaving nucleus
● Template strand map in eukaryotes, from 5′ to 3′
○ Promoter (CAAT box, TATA box), transcription start site (TSS, +1)
○ 5′ untranslated region (UTR), start codon, exons/introns, stop codon, 3′ UTR (poly(A) signal)
○ Terminator
● Initiation
○ RNA Pol (DNA-dependent RNA Pol), transcription factors (TFs) bind promoter
■ RNA Pol binds Pribnow box (prok.), RNA Pol II binds TATA box (euk., ~ –25)
■ TFs bind CAAT box (euk., ~ –70)
● Elongation: RNA Pol II reads template strand 3′ to 5′, synthesizes pre-mRNA 5′ to 3′
○ No priming (RNA Pol) or Okazaki fragments (reading only the template strand) needed
○ Less accurate than DNA replication
○ Produces heterogeneous nuclear RNA (hnRNA)/pre-mRNA
● Termination: RNA Pol reaches terminator sequence, stops transcribing
○ Prokaryotes
■ Rho factor method
● RNA Pol stalls after reaching terminator
● Rho binds rho utilization site (rut, C-rich) on transcript, travels down the transcript using ATP, unwinds
transcript from template DNA
■ Hairpin method
● Terminator sequence is GC-rich palindrome, followed by chain of A’s
● Transcript self-anneals into hairpin (strong H bonds), followed by chain of A–U’s (weak H bonds)
● NusA binds hairpin, stalls RNA Pol, weak A–U duplex unwinds
 ○ Eukaryotes
■ RNA Pol II transcribes poly(A) signal
■ CPSP (cleavage and polyadenylation specificity factor) and CstF (cleavage stimulation factor) transfer from RNA
Pol II to poly(A) signal transcript
■ Recruited proteins cleave transcript, add poly(A) tail to pre-mRNA
● Post-transcriptional modifications
○ Splicing: remove introns (noncoding), ligate exons (coding)
■ Spliceosome: small nuclear RNA (snRNA) + small nuclear ribonucleoproteins (snRNPs)
● Binds intron, recognizes 5′, 3′ splice sites and brings them closer together
● Exons ligate, intron is excised as lariat
■ Alternative splicing: 1 hnRNA can be spliced in diff. ways to produce diff. proteins
○ 5′ cap: 7-methylguanylate triphosphate (m7Gppp) at 5′ end
■ Capping occurs during transcription
● Remove 1 phosphate from 5′ terminal NTP
● Add GTP backwards → 5′–5′ triphosphate bond + PPi
● Methylate N-7 of Gppp
■ Ribosomal binding site during translation
■ Regulates nuclear export, protects mRNA from degradation in cytoplasm
■ Mitochondrial/chloroplast/prokaryotic mRNA lack 5′ caps
● Some bacteria have other caps
○ Poly(A) tail: chain of A’s at 3′ end
■ Polyadenylation occurs as transcription terminates
■ Protects mRNA from degradation in cytoplasm
● Once in cytoplasm, mRNA starts being degraded 3′ to 5′
● Longer poly(A) tail = longer survival in cytoplasm
■ Regulates nuclear export
■ Prokaryotes: poly(A) tail marks mRNA for degradation (!)
● Export: processed mRNA leaves nucleus thru nuclear pores
7.3. Translation
● Ribosome: rRNA + ribosomal proteins (RPs)
○ Large, small subunits: bind only during translation
■ Prokaryotes: total 70S
● Large (50S): 23 + 5S rRNA
● Small (30S): 16S rRNA
■ Eukaryotes: total 80S (all even numbers for subunits, 1828585 for rRNA components)
● Large (60S): 28 + 5.8 + 5S rRNA
● Small (40S): 18S rRNA
● RNA Pol I transcribes 45S pre-rRNA (28 + 18 + 5.8S) in nucleolus
 ● RNA Pol III transcribes 5S rRNA outside nucleolus
● Initiation
○ Small subunit binds mRNA at 5′ end, initiator tRNA binds start codon
■ Prokaryotes: Shine–Dalgarno sequence in 5′ UTR, fmet-tRNA
■ Eukaryotes: 5′ cap, met-tRNA
○ Small subunit travels down mRNA until it reaches start codon
○ Large subunit binds small subunit thru initiation factors (IFs)
● Elongation: ribosome reads mRNA 5′ to 3′, synthesizes protein N- to C-terminus
○ Ribosome sites: “APE”
■ A site: holds incoming aa-tRNA
● EF-Tu (prok.)/EF-1A (euk.) carries aa-tRNA to A site, uses GTP to dissociate itself
● EF-Ts (prok.)/EF-1B (euk.) releases GDP from EF-Tu/EF-1A
■ P site: holds tRNA carrying polypeptide chain
● Peptidyl transferase: forms peptide bond between A-site AA and P-site polypeptide using GTP
● EF-G (prok.)/EF-2 (euk.) moves tRNA/mRNA down the ribosome
■ E site: uncharged tRNA exits ribosome
○ Signal sequence: short N-terminus peptide, indicates destination for protein
■ Secretory, membrane, lysosomal proteins
● Ribosome migrates, attaches to ER
● Signal peptidase removes signal sequence, translation continues
● Protein is glycosylated in ER, transferred to Golgi apparatus, exported
● Termination: release factor (RF) binds stop codon
○ No aa-tRNA exists for stop
○ Peptidyl transferase, termination factors hydrolyze polypeptide from tRNA
○ Ribosomal subunits dissociate
● Post-translational modifications (euk.)
○ Chaperones: ensure correct protein folding
■ Proteins can be misfolded, native state is not always the lowest-energy
○ Cleavage: removes signal sequence, cleaves zymogens, cleaves polyproteins (in prok.)
○ 4° interactions
○ Phosphorylation: by kinases, de/activates protein
○ Carboxylation: adds CA groups to be Ca2+-binding sites
○ Glycosylation: adds oligosaccharides, indicates cellular destination
○ Prenylation: adds lipid groups to membrane-bound enzymes
7.4. Prokaryotic Gene Regulation
● Operon: gene cluster governed by 1 promoter, transcribed on 1 mRNA
● Jacob–Monod model (from upstream to downstream)
○ Regulator: encodes repressor
 ○ Promoter: binds RNA Pol
○ Operator: binds repressor
○ Structural gene: encodes actual protein
● (+) control: protein binds DNA → transcription ↑
● (–) control: protein binds DNA → transcription ↓
● Inducible systems: expression is off by default
○ Default: repressor is bound to operator, blocks RNA Pol
○ Inducer binds and removes repressor, RNA Pol can proceed
○ e.g., lac operon: encodes most lactase when high [lactose], low [glucose]
■ Using lactose is more expensive than using glucose
■ Lactose is inducer, (–) control
■ Catabolite activator protein (CAP): transcriptional activator, (+) control
● Glucose ↓ → cAMP ↑, cAMP binds CAP, CAP binds promoter
■ High [lactose], low [glucose]: most expression
■ High [lactose], high [glucose]: basal (low) expression
■ Low [lactose]: no expression
● Repressible systems: expression is on by default
○ Default: free repressor can’t bind operator, RNA Pol can proceed
○ Corepressor binds repressor, complex binds operator, RNA Pol is blocked
■ (–) feedback: final product = corepressor
○ e.g., trp operon: synthesizes most Trp when low [Trp]
■ Trp is corepressor, (–) control
■ Low [Trp]: expression
■ High [Trp]: no expression, (–) feedback
7.5. Eukaryotic Gene Regulation
● Transcription factors (TFs): transcriptional activators
○ DNA-binding domain: binds TF-binding site (TFBS) in response element (promoter/enhancer)
○ Activation domain: binds other TFs, RNA Pol, histone acetylases, etc.
○ Signal molecules: steroid hormones/2nd messengers bind nuclear receptors, which act as TFs
● Gene amplification: amplifies expression from basal (default, low) levels
○ Enhancers: TFBSs very far away from TSS (~1,000 bp away), either upstream or downstream
■ DNA bends to bring enhancer-bound TFs and promoter-bound TFs together
○ Insulators: block enhancers by changing DNA’s 3D structure
■ Bound by CCCTC-binding factors (CTCFs, a kind of TF)
■ Methylating insulators blocks CTCF-binding, disables insulators
○ Gene duplication: copies gene in series/parallel on same chromosome
● Chromatin remodeling
○ Heterochromatin: tightly coiled, inaccessible to TFs, inactive
 ○ Euchromatin: loosely coiled, TFs can bind, active
○ Histone acetylation: acetylates histone Lys, opens chromatin
■ Acetylated Lys is no longer (+)-charged, weaker DNA–histone interactions
■ Histone de/acetylases
○ DNA methylation: methylates C’s in CpG (and A’s), silences gene
■ Methyl groups block TF-binding or recruit transcriptional repressors
■ 5-methylcytosine (m5C) can spontaneously deaminate into T

8. Membranes
8.1. Fluid Mosaic Model
● Plasma membrane: semipermeable phospholipid bilayer
○ Bilayer: double layer of amphipathic phospholipids
○ Selective permeability: small nonpolar neutral molecules can diffuse thru membrane
■ Others need channels/carriers
○ Glycoprotein coat: carbs associated w/ membrane proteins
■ Cell wall: plants, bacteria, fungi
● Fluid mosaic: phospholipids freely, rapidly move within layer by diffusion
○ Lipid rafts: collections of similar lipids, attachment points for other molecules
■ Involved in signaling
○ Flippases: flip phospholipids between layers
■ Unfavorable, since polar head must cross nonpolar interior of membrane
8.2. Components
● Lipids
○ Glycerophospholipids: triacylglycerols w/ 2 FAs + 1 phosphate group, amphipathic
■ Spontaneous assembly into micelles (monolayer), liposomes (bilayer)
■ Unsaturated FAs: liquid at room temp, fluidity ↑
● Most cannot be synthesized, obtained thru diet
■ Saturated FAs: solid at room temp, fluidity ↓
○ Sphingolipids: sphingosine (or sphingosine deriv.) backbone, amphipathic
■ Ceramides (H), sphingomyelins (phosphatidylcholine/ethanolamine), cerebrosides (sugar), gangliosides
(polysaccharide)
○ Cholesterols: stabilize membranes, amphipathic
■ Low temps: prevents crystalline packing between phospholipids, fluidity ↑
■ High temps: hinders phospholipid movement, fluidity ↓
■ Cell membranes have high cholesterol:phospholipid ratios to ensure fluidity
 ● ~20% of membrane by mass, ~50% of membrane by amount
■ Steroid precursors
○ Waxes: long-chain FA + long-chain alcohol, extremely hydrophobic
■ Stability, rigidity ↑ in interior of membranes
■ Rare in animal membranes
● Proteins
○ Integrated: associated w/ interior of cell membrane
■ Transmembrane: pass completely thru bilayer (e.g., transporters, channels, receptors)
● Receptors: ligand-gated channels, signal transduction (e.g., GPCRs), etc.
■ Embedded: associated w/ cytoplasmic or extracellular surface only
○ Membrane-associated/peripheral
■ Electrostatically bound to bilayer (lipid rafts), bound to other proteins, etc.
● Carbs: cell signaling, recognition (e.g., ABO antigens)
○ Attached to glycoproteins, glycolipids
● Cell junctions: direct intercellular communication
○ Cell adhesion molecules (CAMs): allow cell recognition, help proper differentiation/development
○ Gap junctions/connexons: direct cell–cell communication
■ Connexin hexamers
■ Water, some solutes can pass through
■ e.g., electrical junctions, intercalated discs of cardiac muscle
○ Tight junctions: impermeable (prevent paracellular transport), physical links between epithelial cells
■ Continuous bands around cell to ensure tight seal
○ Desmosomes: bind adjacent cells by anchoring their cytoskeletons together
■ Transmembrane proteins (cadherins) associated w/ intermediate filaments (keratin)
■ Found in interfaces between epithelial layers
■ Hemidesmosomes: attach epithelial cells to basement membrane
8.3. Membrane Transport
● Passive transport: no energy needed (spontaneous, ΔG < 0), driven by ΔS > 0
○ Simple diffusion: particles move down concentration gradient thru membrane
■ Small nonpolar neutral molecules only
■ Osmosis: diffusion of water
● Water moves from low [solute] to high [solute] (a.k.a. from high [water] to low [water])
● Tonicity
○ Hypotonic: [intracellular] < [extracellular], water moves into cell
■ Lysis (animals), turgid (plants)
○ Isotonic: [intracellular] = [extracellular], no net water movement
○ Hypertonic: [intracellular] > [extracellular], water moves out from cell
■ Crenation (animals), plasmolysis (plants)
 ● Osmotic pressure: Π = iMRT, where i = van ’t Hoff factor
○ Hydrostatic pressure exerted on lower-conc. compartment, opposes further influx of water
○ “Sucking” pressure: draws water into cell in proportion to [soln.]
○ Colligative property: depends only on [solute], not on the identity of those solutes
○ Facilitated diffusion: diffusion of impermeable particles
■ Large, polar or charged molecules
■ Carriers: open to only 1 side of membrane at a time
● Substrate binds carrier, carrier changes conformation, substrate dissociates into cell
○ Occluded state: carrier is closed to both sides during conformational change
■ Channels: open to both sides of membrane
● Closed, inactivated states
● Active transport: requires energy (nonspontaneous, ΔG > 0)
○ Particles move against concentration gradient
○ Primary: uses energy (ATP hydrolysis) to power transport directly
■ Transmembrane ATPases
○ Secondary/coupled: harnesses energy of another particle moving down its gradient
■ Symport/cotransport: both particles flow in same direction
● e.g., Na+/glucose cotransporter in small intestine, kidney proximal tubules
■ Antiport/exchange: particles flow in opposite directions
● Endocytosis: plasma membrane invaginates, engulfs material in vesicle
○ Certain membrane receptors trigger endocytosis, vesicle-coating proteins (clathrin) invaginate membrane
○ Pinocytosis: fluids, dissolved particles
○ Phagocytosis: large solids (e.g., bacteria)
● Exocytosis: secretory vesicle fuses w/ plasma membrane, material is released
○ e.g., neurotransmitter release from synaptic vesicles into synaptic cleft
8.4. Specialized Membranes
● Membrane potential (Vm): diff. in electrical potential across membrane
○ Electrochemical (EC) gradient: forms from ion impermeability and ion-channel selectivity
○ Resting potential: –40 to –80 mV
○ Maintaining membrane potential requires energy
■ Leak channels: ions passively diffuse through membrane
● K+ leaks out over time: cell membranes are more permeable to K+
○ # K+ leak channels ≫ # Na+ leak channels
■ Na+/K+ pump (Na+/K+-ATPase): maintains low [Na+], high [K+] inside cell
● Uses energy to transport 3 Na+ out, 2 K+ in
● Net –1 charge each time
○ Nernst equation: Eion = RT/(zF) ln [outside]/[inside], where z = charge of ion, F = Faraday constant
■ At body temp, Eion ≈ (62 mV)/z log [outside]/[inside]
 ■ Goldman–Hodgkin–Katz voltage equation:
● Vm = (62 mV) log (PNa+[outside] + PK+[outside] + PCl–[inside])/(PNa+[inside] + PK+[inside] + PCl–[outside]),
where P = permeability of ion
○ Note: inside/outside for Cl– is reversed, b/c Cl– is (–)-charged
● Mitochondrial membranes
○ Outer membrane: large pores, highly permeable (ions, small proteins)
■ Encloses intermembrane space
○ Inner membrane: much less permeable, cristae (infoldings) increase SA
■ Encloses mitochondrial matrix: citric acid cycle
■ High [cardiolipin], no cholesterols in membrane

9. Glycolysis, Gluconeogenesis, Pentose Phosphate Pathway

9.1. Glucose Transporters
● GLUT1: transporter in RBCs, blood–brain barrier, placenta
● GLUT2: low-affinity (high Km) transporter in liver, pancreatic β cells, kidney prox. tubules, small intestine
○ Doesn’t respond to insulin
○ 1st-order kinetics: rate of uptake ∝ [glucose]
○ In liver cells: captures, stores excess glucose in hepatic portal vein after a meal
■ If [glucose] < Km, then most glucose bypasses liver, enters peripheral circulation
○ In pancreatic β cells: acts as glucose sensor
● GLUT3: very high-affinity (very low Km) transporter in neurons, sperm
● GLUT4: high-affinity (low Km ≈ [blood sugar]), insulin-dependent transporter in fat, muscle
○ Insulin → more GLUT4 on surface, uptake Glucose faster
○ Zero-order kinetics: constant uptake, independent of [glucose]
■ Transporter is saturated slightly above [blood glucose]
○ In muscles: excess glucose → glycogen
○ In fat: excess glucose → … → dihydroxyacetone phosphate (DHAP)
■ DHAP + NADH ⥫(glycerol 3-P DH (GPDH))⥬ glycerol 3-P + NAD + → … → triacylglycerols

9.2. Glycolysis
● Breaks down glucose into 2 pyruvate, producing some energy
● Occurs in cytoplasm
● Net: glucose + NAD+ + 2 ADP + 4 Pi → 2 pyruvate + 2 NADH + 2 ATP
● Steps
○ Glucose (Glc) + ATP ⤚(hexokinase)→ Glc 6-P (G6P) + ADP
■ Traps Glc from leaking out thru GLUT’s
 ■ Irreversible rxn.: attaching phosphate requires energy, but some energy from ATP hydrolysis is wasted
■ Hexokinase: in most tissues
● Lower Km (need Glc for energy), lower vmax (saturated even during fasting)
● Inhibited by G6P (enough Glc in glycolysis)
● Hexokinase has a much higher affinity for glucose than its isozyme, glucokinase
■ Glucokinase: in liver, pancreatic β islets
● Higher Km (save Glc from energy-storage pathways), higher vmax (quickly adjust blood sugar)
● Induced by insulin
○ G6P ⥫(G6P isomerase)⥬ Fru 6-P (F6P)
■ Sets up aldol cleavage later
○ F6P + ATP ⤚(phosphofructokinase-1 (PFK-1))→ Fru 1,6-bisP (F1,6BP) + ADP
■ Rate-limiting, irreversible rxn.: commits Glc to oxidation
■ Main control point in glycolysis
● Activated by AMP: not enough ATP in cell
● Inhibited by ATP, citrate: enough energy in cell
● In liver: hormonal control thru PFK-2
○ PFK-2 converts a tiny amount of F6P to F2,6BP, which activates PFK-1
○ Activated by insulin, inhibited by glucagon
○ Overrides (–) feedback from ATP, excess glycolysis products are stored as glycogen/FAs/etc.
○ F1,6BP ⥫(aldolase A)⥬ glyceraldehyde 3-P (GAP) + dihydroxyacetone phosphate (DHAP)
■ Splits hexose into 2 trioses
○ DHAP ⥫(triose phosphate isomerase (TPI))⥬ GAP
■ Only GAP can proceed in glycolysis
○ GAP + NAD+ + Pi ⥫(GAPDH)⥬ 1,3-bisphosphoglycerate (1,3BPG) + NADH
■ Oxidizes GAP, reduces NAD+, excess energy is used to attach a phosphate
● NADH is oxidized for energy (aerobic) or for Le Châtelier’s principle (anaerobic)
■ Produces unstable anhydride
○ 1,3BPG + ADP ⥫(phosphoglycerate kinase (PGK))⥬ 3-phosphoglycerate (3PG) + ATP
■ Cleaves anhydride, uses energy to form ATP
○ 3PG ⥫(phosphoglycerate mutase (PGM))⥬ 2PG
○ 2PG ⥫(enolase)⥬ phosphoenolpyruvate (PEP) + H2O
■ Dehydration creates high-energy trapped enol
● Cannot tautomerize to more stable keto, b/c O is bound to phosphate
○ PEP + ADP ⤚(pyruvate kinase (PK))→ pyruvate + ATP
■ Irreversible rxn.
■ Feedforward activation: activated by F1,6BP
● Fermentation
○ In anaerobic respiration, Le Châtelier’s principle stops/reverses glycolysis
 ■ Pyruvate, NADH build up
■ To drive glycolysis, pyruvate is reduced as waste, NADH is reoxidized
○ Lactic acid fermentation in humans
■ Pyruvate + NADH ⥫(lactate DH)⥬ lactate + NAD+
○ Ethanol fermentation in yeast
■ Pyruvate ⥫(pyruvate decarboxylase)⥬ acetaldehyde + CO 2
○ Acetaldehyde + NADH ⥫(alcohol DH)⥬ ethanol + NAD +
● Glycolysis products
○ DHAP: used in fat, liver for synthesizing triacylglycerol
■ Reduces to glycerol 3-P, dephosphorylates to glycerol
○ 1,3BPG, PEP: transfer their phosphates to produce ATP
■ Only source of ATP in anaerobic respiration
○ Pyruvate
■ Anaerobic respiration: wasted as lactate
■ Aerobic respiration: enters citric acid cycle as acetyl-CoA
■ Gluconeogenesis: converted to oxaloacetate by pyruvate carboxylase (PC)
● Glycolysis in RBCs
○ RBCs lack mitochondria, anaerobic glycolysis is only source of ATP
○ BPG mutase (BPGM): isomerizes 1,3BPG to 2,3BPG
■ 2,3BPG binds β chains of hemoglobin A (HbA), oxygen affinity ↓
● Does not bind fetal hemoglobin (HbF): allows fetus to get oxygen thru placenta
■ Unloads more oxygen during exercise
9.3. Other Monosaccharides
● Galactose (Gal) metabolism: turns Gal into Glc 1-P
○ Net: Gal + ATP → Glc 1-P + ADP
○ Steps
■ Lactose ⤚(lactase)→ Glc, Gal
■ Gal + ATP ⤚(galactokinase)→ Gal 1-P + ADP
● Traps galactose in cell
■ Gal 1-P + UDP-Glc ⥫(Gal 1-P uridylyltransferase (GALT)) ⥬ Glc 1-P (G1P) + UDP-Gal
■ UDP-Gal ⥫(epimerase)⥬ UDP-Glc
● Epimerases: converts 1 sugar epimer to another
■ Glucose enters its metabolism
○ Diseases
■ Lactose intolerance: no lactase
● 1°: hereditary
● 2°: damage to intestinal lining containing lactase
● Symptoms are from bacterial lactose fermentation → methane, H 2, small organic acids
 ■ Galactosemia: no galactokinase or GALT
● Gal ⤚(aldose reductase)→ galactitol
● Galactitol buildup in lens causes osmotic damage, cataracts
● Fructose (Fru) metabolism: turns Fru into glycolysis intermediates
○ Net: Fru + 2 ATP → 2 GAP + 2 ADP
○ Steps
■ Sucrose ⤚(sucrase)→ Glc, Fru
■ Fru + ATP ⤚(fructokinase)→ Fru 1-P + ADP
■ Fru 1-P ⥫(aldolase B)⥬ DHAP + glyceraldehyde
■ Glyceraldehyde + ATP ⤚(triokinase)→ GAP + ADP
■ DHAP, GAP enter glycolysis
9.4. Pyruvate Dehydrogenase
● Found in mitochondrial matrix
● Net: pyruvate + CoASH + NAD+ → acetyl-CoA + CO2 + NADH
● Steps
○ E1: pyruvate DH (PDH)
■ Decarboxylates pyruvate, stabilizes resulting carbanion w/ thiamine PPi (TPP) and Mg2+
■ Oxidizes carbonyl (into basically CA), reduces lipoamide, and attaches the resulting acetyl to it
○ E2: dihydrolipoyl transacetylase
■ Transfers acetyl to CoA, storing redox energy in thioester
○ E3: dihydrolipoamide DH
■ Oxidizes and restores lipoamide, shuttling its 2 e– to FAD
■ Shuttles 2 e– from FADH2 to NAD+
● Regulation
○ Activated by insulin in liver
○ Inhibited by ATP, NADH, acetyl-CoA
○ PDH kinase: phosphorylates, inhibits PDH in high [ATP]
○ PDH phosphatase: dephosphorylates, activates PDH in high [ADP]
9.5. Glycogen
● Glycogen: glucose storage, stored in cytoplasm as granules
○ Synthesized/degraded in liver, skeletal muscles
● Glycogenesis
○ Net: Glc + ATP + UTP → glycogen + ADP + UDP + PP i
○ Steps
■ Glucose + ATP ⤚(glucokinase)→ G6P + ADP
■ G6P ⥫(phosphoglucomutase)⥬ G1P
■ G1P + UTP → UDP-Glc + PPi
 ● Activates Glc to allow integration into glycogen
■ UDP-Glc ⤚(glycogen synthase)→ glycogen + UDP
● Glycogen synthase: forms α-1,4 glycosidic bonds, rate-limiting
○ Activated by G6P, insulin
○ Inhibited by Epi, glucagon
● Branching enzyme: adds α-1,6-linked branches
○ Breaks α-1,4 bond
○ Moves free oligoglucose, forms α-1,6 bond
● Glycogenolysis
○ Net: glycogen → Glc
○ Steps
■ Glycogen + Pi ⤚(glycogen phosphorylase)→ G1P
● Glycogen phosphorylase: breaks α-1,4 glycosidic bonds, rate-limiting
○ Activated by glucagon (liver), Epi, AMP (skeletal muscle)
● Debranching enzymes
○ Breaks α-1,4 bond adjacent to branch point
○ Moves free oligoglucose to exposed end, forms α-1,4 bond
○ Breaks α-1,6 bond, releasing free Glc
■ G1P ⥫(phosphoglucomutase)⥬ G6P
■ G6P ⤚(G6Pase)→ glucose + Pi
● von Gierke’s disease: G6Pase defect
○ No gluconeogenesis, need constant supply of carbs
○ G6P buildup in liver → liver damage

9.6. Gluconeogenesis
● Liver (and kidneys) synthesize glucose during fasting
○ Activated by Epi, glucagon
○ Inhibited by insulin
● Sources (G3OL)
○ Oxaloacetate from citric acid cycle (cataplerosis)
■ Oxaloacetate is reduced to malate (oxaloacetate can’t leave mitochondria)
■ Malate–Asp shuttle: transports malate into cytoplasm, antiport w/ α-ketoglutarate
■ Malate is oxidized back to oxaloacetate
○ Galactose, fructose metabolism
○ Lactate from anaerobic glycolysis
■ Lactate ⥫(lactate DH)⥬ pyruvate
○ Glycerol 3-P from triacylglycerols
■ Glycerol 3-P + NAD+ ⥫(GPDH)⥬ DHAP + NADH
■ DHAP enters reversed glycolysis
 ○ Glucogenic AAs
■ Ala + α-ketoglutarate (α-KG) ⥫(Ala aminotransferase)⥬ pyruvate + Glu
■ etc.
● Net: pyruvate + bicarbonate + ATP + GTP → Glc + ADP + GDP + 2 P i + CO2
● Steps
○ Pyruvate + bicarbonate + ATP ⤚(PC)→ oxaloacetate + ADP
■ Activated by acetyl-CoA
● This acetyl-CoA must come from β-oxidation of FAs
■ Biotin w/ Lys side chain
○ Oxaloacetate + GTP ⤚(PEP carboxykinase (PEPCK))→ PEP + GDP + CO 2
■ Activated by cortisol, glucagon
○ PEP ⇌ … ⇌ F1,6BP
○ F1,6BP ⤚(F1,6BPase)→ F6P + Pi
■ Irreversible (not coupled to anything, energy wasted), rate-limiting step
■ Activated by ATP
■ Inhibited by AMP, F2,6BP
○ F6P ⇌ G6P
○ G6P ⤚(G6Pase)→ glucose + Pi
■ Found only in ER lumen of liver cells
● G6P is transported into ER, glucose is transported out of cell
● No G6Pase in skeletal muscle: glycogen is burned within muscle only, not exported
○ Glycolysis can occur in all cells, while gluconeogenesis cannot
9.7. Pentose Phosphate Pathway
● Pentose phosphate pathway (PPP)/hexose monophosphate (HMP) shunt
○ Occurs in cytoplasm of all cells
○ Produces NADPH: e– donor, reducing agent in rxns.
■ FA/cholesterol synthesis
■ Bleach synthesis in WBCs: bactericide
■ Glutathione synthesis: antioxidant
● G6PDH deficiency: no PPP → no glutathione → more oxidative stress
○ Produces ribose 5-P: for nucleotides
● Glucose → G6P
● G6P + H2O + NADP+ ⤚(G6P dehydrogenase (G6PDH))→ 6-phosphogluconate + H+ + NADPH
○ Rate-limiting step
○ Activated by NADP+, insulin
○ Inhibited by NADPH
● 6-phosphogluconate + NADP+ → ribulose 5-P + NADPH + CO2
● Ribulose 5-P ⇌ ribose 5-P ⇌ [a bunch of 4–7 C sugars]
 ○ Equilibrated pool of sugars for biosynthesis
■ e.g., ribose 5-P is used for making RNA/DNA

10. Aerobic Respiration

10.1. Sources of Acetyl-CoA
● Pyruvate dehydrogenase (PDH) complex
○ Found in mitochondrial matrix
○ Net: pyruvate + CoASH + NAD+ → acetyl-CoA + CO2 + NADH
○ Steps
■ E1: PDH
● Decarboxylates pyruvate, stabilizes resulting carbanion w/ thiamine PPi (TPP) and Mg2+
● Oxidizes carbonyl, reduces lipoamide, and attaches the acetyl to it
■ E2: dihydrolipoyl transacetylase
● Transfers acetyl to CoA, storing redox energy in thioester
■ E3: dihydrolipoamide DH
● Oxidizes and restores lipoamide, shuttling its 2 e– to FAD
● Shuttles 2 e– from FADH2 to NAD+
○ Regulation
■ Inhibited by ATP, NADH
■ PDH kinase: phosphorylates, inhibits PDH in high [ATP]
■ PDH phosphatase: dephosphorylates, activates PDH in high [ADP]
● β-oxidation
○ FA + ATP + CoASH → acyl-CoA + AMP + PPi
■ Activates FA
○ Acyl-CoA ⇌ acyl-carnitine + CoASH
■ Transesterification: FA-carnitine shuttles into mitochondrion, acyl-CoA re-forms
○ Mitochondrial acyl-CoA is β-oxidized into acetyl-CoA’s
● AA catabolism
○ Transamination: ketogenic AAs ⇌ ketone bodies
● Ketone bodies
○ Formed in liver, converted into acetyl-CoA
○ Main energy source for brain during starvation
● Alcohol
○ Ethanol ⥫(alcohol DH)⥬ acetaldehyde ⤚(acetaldehyde DH)→ acetate
○ NADH also builds up → citric acid cycle ↓
 ○ Acetyl-CoA from alcohols goes into FA synthesis
10.2. Citric Acid Cycle
● Citric acid/Krebs/tricarboxylic acid (TCA) cycle: fully oxidizes acetyl-CoA, produces e– carriers (NADH, QH2)
● Occurs in mitochondrial matrix
● Net: acetyl-CoA + 3 NAD+ + Q + GDP → 2 CO2 + 3 NADH + QH2 + GTP
○ Requires oxygen to proceed: w/o oxygen, NADH accumulates from e– transport chain (ETC), stalls cycle
● Steps
○ Acetyl-CoA + oxaloacetate + H2O ⤚(citrate synthase)→ citrate + CoASH + H+
■ ΔG ≪ 0: irreversible rxn., drives cycle forward
■ Inhibited by ATP, NADH, citrate, succinyl-CoA
○ Citrate ⥫(aconitase)⥬ cis-aconitate + H2O ⥫(aconitase)⥬ isocitrate
■ Isomerizes citrate to isocitrate
■ Aconitase requires Fe2+ cofactor
○ Isocitrate + NAD+ ⥫(isocitrate DH)⥬ oxalosuccinate + NADH ⇌ α-ketoglutarate (α-KG) + CO 2 + NADH
■ 1st redox, rate-limiting step of citric acid cycle
■ Inhibited by ATP, NADH
■ Activated by ADP, NAD+
○ α-KG + CoASH + NAD+ ⤚(α-KG DH)→ succinyl-CoA + CO2 + NADH
■ Uses excess redox energy to create thioester (high-energy), irreversible
■ α-KG DH coenzymes, mechanism are same as PDH
■ Inhibited by ATP, NADH, succinyl-CoA
■ Activated by ADP, Ca2+
○ Succinyl-CoA + GDP + Pi ⥫(succinyl-CoA synthetase)⥬ succinate + GTP + CoASH
■ Spends energy stored in thioester to create GTP
■ Nucleoside diphosphate kinase eventually transfers phosphate from GTP to ATP
○ Succinate + Q ⥫(succinate DH)⥬ fumarate + QH 2
■ 2nd redox, occurs on inner membrane
■ Succinate DH: flavoprotein, bonded to e– acceptor FAD
● Complex II in ETC
○ Fumarate + H2O ⥫(fumarase)⥬ malate
○ Malate + NAD+ ⥫(malate DH)⥬ oxaloacetate + NADH
■ 3rd redox
■ Regenerates oxaloacetate
● Where did oxaloacetate C’s go?
○ Carbonyl C became one of the backbone C’s
○ CH2 C became one of the CAs
○ 2 CA’s were lost as CO2
● Where did acetyl-CoA C’s go?
 ○ CH3 C became one of the backbone C’s
○ Thioester C became one of the CAs
● Net results of aerobic respiration
○ Metabolic steps
■ Glycolysis: glucose + 2 NAD+ + 2 ADP → 2 pyruvate + 2 NADH + 2 ATP
■ PDH: 2× (pyruvate + CoASH + NAD+ → acetyl-CoA + CO2 + NADH)
■ Citric acid cycle: 2× (acetyl-CoA + 3 NAD+ + Q + GDP → 2 CO2 + CoASH + 3 NADH + QH2 + GTP)
○ ATP yield
■ 1 GTP = 1 ATP
■ 1 NADH ≈ 2.5 ATP, on average
■ 1 QH2 ≈ 1.5 ATP
■ Each glucose yields 30–32 ATP aerobically
10.3. Electron Transport Chain
● Redox to get from NAD+/NADH (lowest red. potential) to O2/H2O (highest red. potential)
● Proton-motive force (PMF): electrochemical gradient
○ Stores energy, couples e– transport (exergonic) to oxidative phosphorylation (endergonic)
○ Intermembrane space: high [H+], (+)-charged, low pH
○ Matrix: low [H+], (–)-charged, high pH
● Occurs across mitochondrial cristae of inner membrane
● e– carriers: NAD+/NADH, FMN/FMNH2, succinate/fumarate, FAD/FADH2, ubiquinone/ubiquinol (Q/QH2), FeSox/red, cyt cox/red,
O2/H2O
● Steps
○ Complex I: NADH:Q oxidoreductase
■ Net: NADH + Q + 5 H+in ⇌ NAD+ + QH2 + 4 H+out
■ Shuttles 2 e– from NADH to FMN to FeS clusters to Q
○ Complex II: succinate:Q oxidoreductase, a.k.a. succinate DH
■ Net: succinate + Q ⇌ fumarate + QH2
■ Shuttles 2 e– from succinate to FAD to FeS cluster to Q
■ No coupled H+ transport, b/c red. potentials of succinate/fumarate, Q/QH2 are similar
○ Complex III: Q:cyt c oxidoreductase
■ Net: QH2 + 2 cyt cox + 2 H+in ⇌ Q + 2 cyt cred + 4 H+out
■ Shuttles 2 e– from QH2 to FeS cluster to 2 cyt cox
■ Q cycle
● QH2 + Q + cyt cox ⇌ Q + Q• + cyt cred + 2 H+out
○ QH2 pumps 2 H+ out, gives 1 e– each to cyt cox and Q
● QH2 + Q• + cyt cox + 2 H+in ⇌ Q + QH2 + cyt cred + 2 H+out
○ QH2 pumps 2 H+ out, gives 1 e– each to cyt cox and Q• (semiquinone)
○ Complex IV: cyt c oxidase
 ■ Net: 2 cyt cred + ½ O2 + 4 H+in ⇌ 2 cyt cox + H2O + 2 H+out
■ Shuttles 2 e– from 2 cyt cred to cyt oxidase to ½ O2 (final e– acceptor)
● NADH shuttles: NADH from glycolysis can’t enter mitochondria
○ Glycerol 3-P shuttle: converts NADH to QH2, 1.5 ATP per NADH
■ Glycerol 3-P can enter mitochondria
● Cytosol: DHAP + NADH ⥫(GPDH-C)⥬ glycerol 3-P + NAD+
● Outer side of inner mitochondrial membrane: glycerol 3-P + Q ⥫(GPDH-M) ⥬ DHAP + QH 2
○ Malate–Asp shuttle
■ Convert oxaloacetate into malate, which can cross mitochondria
● Cytosol: oxaloacetate + NADH ⥫(malate DH)⥬ malate + NAD +
● Matrix: malate + NAD+ ⥫(malate DH)⥬ oxaloacetate + NADH
■ Regenerate oxaloacetate from Asp, which can cross mitochondria
● Matrix: oxaloacetate + Glu ⥫(Asp transaminase)⥬ Asp + α-KG
● Cytosol: Asp + α-KG ⥫(Asp transaminase)⥬ oxaloacetate + Glu

10.4. Oxidative Phosphorylation

● Releases energy stored in electrochemical gradient for ATP synthesis
● Chemiosmotic coupling: PMF → mechanical energy → chemical energy
● ATP synthase: reduces oxygen in the final step of the electron transport chain in order to utilize the proton gradient and generate
ATP
○ FO region: ion channel for H+, w/in inner mitochondrial membrane (hydrophobic)
■ c subunits: 10 H+ needed to turn rotor in humans
○ F1 region: catalyzes ADP + Pi ⇌ ATP, protrudes into matrix (hydrophilic)
■ α/β hexamer: fits 3 ADP, each c10 turn catalyzes 1 ATP synthesis
● Respiratory control: regulates ox. phos.
○ Abundant oxygen: rate of ox. phos. depends on [ADP]
■ ADP activates isocitrate DH → faster citric acid cycle → [NADH], [QH2] ↑ → faster ETC
○ Limited oxygen: slower ox. phos.
■ Slower ETC → [NADH], [QH2] ↓ → citric acid cycle inhibited

11. Lipid, Amino Acid Metabolism

11.1. Lipid Digestion, Absorption
● Digestion
○ Dietary fat: mostly triacylglycerols (TGLs), some cholesterols/cholesteryl esters/phospholipids/free FAs
○ Emulsified in duodenum w/ water, bile (secreted by liver)
■ Small fat clusters, SA ↑
 ○ Hydrolyzed into 2-monoacylglycerols, free FAs, cholesterols
■ By pancreatic lipase, colipase, cholesterol esterase (secreted by lipase)
● Micelle formation
○ 2-monoacylglycerols, free FAs, cholesterols, bile salts form micelles
○ Bile salts are reabsorbed at end of ileum
○ Remaining fats are indigestible, excreted
● Adsorption
○ Micelles adsorb into brush border of intestinal mucosa
○ Lipids re-esterify into TGLs/cholesteryl esters
○ Packaged into chylomicrons w/ apoproteins, fat-soluble vitamins, other lipids
○ Exported into lymphatic system thru lacteals, enter bloodstream thru thoracic duct
■ Short-chain FAs are more water-soluble, just diffuse into bloodstream
11.2. Lipid Mobilization
● Hormone-sensitive lipase (HSL) in adipose: hydrolyzes TGLs into glycerol, free FAs
○ Activated by insulin ↓, cortisol, Epi
● Lipoprotein lipase (LPL) in liver: metabolizes chylomicrons, very-low-density lipoproteins (VLDLs)
11.3. Lipid Transport
● Lipoproteins: apolipoproteins + lipids, allow hydrophobic FAs to be transported thru bloodstream
● Lipoprotein density: protein:lipid ratio
○ Chylomicrons: deliver digested TGLs, cholesteryl esters from intestine to cells
■ Very water-soluble, built in intestinal lining
○ Very-low-density (VLDLs): deliver TGLs, cholesterols, FAs from liver to cells
■ Built in liver
○ Intermediate-density (IDLs)/VLDL remnants: VLDLs that have deposited TGLs in adipose
■ Some are reabsorbed by liver
■ Some pick up HDL cholesteryl esters to form LDLs
○ Low-density (LDLs): deliver cholesterol to cells
■ Cholesterol: used for biosynthesis, plasma membranes, bile acid/salt synthesis, steroidogenesis
■ “Bad” cholesterol, for carrying cholesterol
○ High-density (HDLs): collect excess cholesterol in blood (cholesterol recovery)
■ Also deliver cholesterol to steroidogenic cells
■ Also transfer apolipoproteins to other lipoproteins
■ Built in liver, intestines
■ “Good” cholesterol, for recovering cholesterol for excretion
● Apolipoproteins: receptor proteins in lipoproteins, used for signaling
○ apoA-I: activates lecithin–cholesterol acyltransferase (LCAT), which catalyzes cholesterol esterification
○ Apolipoproteins B: primary apolipoprotein
 ■ apoB-48: mediates chylomicron secretion
■ apoB-100: allows LDL uptake by liver
○ apoC-II: activates LPL
○ apoE: allows chylomicron remnant/VLDL uptake by liver
11.4. Cholesterol Metabolism
● Cholesterol sources
○ LDL, HDL
○ Biosynthesis in liver
■ Citrate shuttle exports mitochondrial acetyl-CoA into cytoplasm
■ 2 acetyl-CoA ⥫(thiolase)⥬ acetoacetyl-CoA + CoASH
● Claisen condensation: enolized thioester attacks carbonyl of another thioester
■ Acetoacetyl-CoA + acetyl-CoA ⥫(HMG-CoA synthase)⥬ 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) +
CoASH
■ HMG-CoA + 2 NADPH ⤚(HMG-CoA reductase)→ mevalonate + 2 NADP +
● HMG-CoA reductase aids in the production of cholesterol in the smooth ER
● Rate-limiting step, occurs in smooth ER
● NADPH comes from PPP
■ Mevalonate → … → cholesterol, w/ NADPH as reducing agent
● Activated by insulin
● Inhibited by cholesterol
● Lecithin–cholesterol acyltransferase (LCAT): adds FA to cholesterol to produce cholesteryl ester (water-soluble)
○ Found in bloodstream
○ Activated by HDL apoproteins
● Cholesteryl ester transfer protein (CETP): transfers cholesteryl esters from HDL to IDL, forming LDL
11.5. Fatty Acids, Triacylglycerols
● Naming
○ (# C’s):(# double bonds)
○ ω numbering system: position of last double bond, counted from end
● Saturated: no double bonds
○ Palmitic acid (16:0): only FA that can be biosynthesized de novo in humans
● Unsaturated: double bonds
○ Most unsaturated FAs can’t be biosynthesized in humans, must come from diet
■ Most natural unsaturated FAs are cis
■ Trans fats are unsaturated but solids at room temp, membrane fluidity ↓
○ Linoleic acid: ω-6 FA (18:2 cis-9,12)
○ α-linoleic acid: ω-3 FA (18:3 cis-9,12,15)
● De novo FA synthesis: creates palmitic acid (16:0) from acetyl-CoA
 ○ Nontemplate synthesis: no nucleic template needed
○ Occurs mostly in liver (and some in fat)
○ Activated by insulin after high-carb meal
○ Steps
■ PDH produces acetyl-CoA from pyruvate in matrix
■ Citrate shuttle exports mitochondrial acetyl-CoA to cytoplasm
● Citrate synthase converts acetyl-CoA + oxaloacetate to citrate
● Citrate lyase reverts citrate to oxaloacetate + acetyl-CoA
● Oxaloacetate returns to mitochondria
■ Acetyl-CoA + bicarbonate + ATP ⤚(acetyl-CoA carboxylase (ACC))→ malonyl-CoA + ADP
● Rate-limiting step, biotin cofactor
● Malonyl-CoA: –OOC–CH2–C(O)SCoA
■ FA synthase/palmitate synthase
● Activate growing chain, malonyl-CoA w/ acyl carrier protein (ACP)
○ Requires pantothenate (vit. B5) cofactor
● ⦚–C–C(O)S–ACP + malonyl-ACP → ⦚–C–C(O)–C–C(O)S–ACP + CO2 + ACP–SH
○ Condensation: deprotonate α-C of malonyl-ACP (1,3-dicarbonyl), Nuc attacks carbonyl of FA chain
● ⦚–C–C(O)–C–C(O)S–ACP + NADPH ⇌ ⦚–C–C(OH)–C–C(O)S–ACP + NADP+
○ Reduce β-carbonyl to hydroxyl
● ⦚–C–C(OH)–C–C(O)S–ACP ⇌ ⦚–C–C=C–C(O)S–ACP + H2O
○ Dehydrate hydroxyl
● ⦚–C–C=C–C(O)S–ACP + NADPH → ⦚–C–C–C–C(O)S–ACP + NADP+
○ Reduce alkene to alkane
● Repeat 8 times
■ Some elongation/desaturation in SER
● Triacylglycerol synthesis: attaches 3 FA-CoA’s to glycerol
○ Occurs mostly in liver (and some in fat)
○ Packaged as VLDLs
● β-oxidation
○ Occurs in mitochondria (< 20 C’s), peroxisomes (> 20 C’s)
■ Long-chain FAs (LCFAs, 14–20 C’s) need carnitine shuttle to enter mitochondria
● Acyl-CoA can’t enter mitochondria, but acyl-carnitine can
● Rate-limiting step of β-oxidation
○ Activated by glucagon, inhibited by insulin
○ Steps
■ Activation: FA + CoASH + ATP ⤚(FA-CoA synthetase)→ FA-CoA + AMP + PP i
● Occurs in cytoplasm, traps FA in cell
■ ⥫–C–C–C–C(O)SCoA + Q ⥬(acyl-CoA DH)⥬ ⦚–C–C=C–C(O)SCoA + QH2
 ● Oxidize α-β alkane to alkene
■ ⦚–C–C=C–C(O)SCoA + H2O ⥫(enoyl-CoA hydratase)⥬–C–C(OH)–C–C(O)SCoA
● Hydrate alkene at β-C
■ ⦚–C–C(OH)–C–C(O)SCoA + NAD+ ⥫(hydroxyacyl-CoA DH)⥬ ⦚–C–C(O)–C–C(O)SCoA + NADH
● Oxidize hydroxyl to carbonyl
■ ⤚ –C–C(O)–C–C(O)SCoA + CoASH ⤚ (thiolase)→ ⦚–C–C(O)SCoA + acetyl-CoA
● Split β-keto acid
○ Each 2 C’s yield 1 QH2, 1 NADH
○ β-oxidizing odd-numbered FAs
■ Carboxylate the remaining propionyl-CoA
● Requires biotin (vit. B7) cofactor
■ Isomerize methylmalonyl-CoA into succinyl-CoA
● Requires cobalamin (vit. B12) cofactor
○ Pernicious anemia: B12 deficiency6
■ Succinyl-CoA enters citric acid cycle or gluconeogenesis
○ β-oxidizing unsaturated FAs
■ Double bond at odd-numbered C: just isomerize to move double bond
● cis 3,4 → trans 2,3
● Yields only NADH (no QH2 produced)
■ Double bond at even-numbered C: oxidize to yield QH2, reduce conjugated double bonds using NADPH, then
isomerize to move double bond
● cis 2,3 + cis 4,5 → trans 3,4 → trans 2,3
● Yields QH2 and NADH, but costs NADPH
11.6. Ketone Bodies
● Ketone bodies: acetoacetate, β-hydroxybutyrate
● Metabolism
○ Cardiac/skeletal muscle, renal cortex revert ketone bodies to acetyl-CoA
○ Most energy source for brain during prolonged fasting
■ Ketone body metabolism → acetyl-CoA inhibits PDH → glycolysis, glucose uptake ↓ in brain
■ Conserves proteins, prevents gluconeogenesis from AAs
● Ketogenesis
○ Occurs in liver mitochondria due to acetyl-CoA buildup (from β-oxidation of fats) in fasting
○ 3 acetyl-CoA ⥫(thiolase, HMG-CoA synthase)⥬ HMG-CoA + 2 CoASH
○ HMG-CoA ⥫(HMG-CoA lyase)⥬ acetoacetate + acetyl-CoA
○ Reduce acetoacetate to β-hydroxybutyrate using NADH
○ Acetoacetate → acetone + CO2
■ Non-enzymatic rxn., acetone cannot be metabolized
● Ketolysis
 ○ Re-oxidize β-hydroxybutyrate to acetoacetate using NAD+
○ Activation: acetoacetate + succinyl-CoA ⥫(thiophorase) ⥬ acetoacetyl-CoA + succinate
○ Split acetoacetyl-CoA into 2 acetyl-CoA
11.7. Protein Catabolism
● Last resort: muscle (esp. cardiac) is essential to survival
● Proteolysis from diet
○ Stomach protease: pepsin
○ Pancreatic proteases: trypsin, chymotrypsin, carboxypeptidases A/B
○ Small intestinal brush-border enzymes: dipeptidase, aminopeptidase
● Transamination: AA + KA ⇌ KA + AA
○ Requires PLP (vit. B6) cofactor, which stabilizes intermediate carbanion
○ Pairs: Ala/pyruvate, Asp/oxaloacetate, Glu/α-KG, etc.
● Deamination: KA ⇌ AA + NH3
● Urea cycle
○ Most cells waste N as Glu (most transamination)
○ Muscles waste N as Ala
○ Deamination wastes N as NH3
○ Liver wastes N as urea: CO(NH2)2
● Glucogenic AAs: can feed into gluconeogenesis
○ All except L’s (Leu, Lys)
● Ketogenic AAs: can be converted into acetyl-CoA, ketone bodies
○ “LFIT”: Leu, Lys, Phe, Iso, Thr, Tyr, Trp

12. Metabolism Bioenergetics, Regulation

12.1. Thermodynamics, Bioenergetics
● Systemic: open system
● Sub/cellular: closed system
● ΔU = Q – W, where U = internal energy, Q = heat
● ΔG = ΔH – TΔS
○ At constant pressure and volume, ΔU = ΔH = Q
● ΔG° = –RT ln K, where K = equilibrium constant
○ ΔG = ΔG° + RT ln Q, where Q = rxn. quotient
■ Standard conds. (ΔG°): 1 M, 1 atm, 25 °C
■ Modified standard state (ΔG°′): standard conds., and pH 7
● ΔGcell° = –nFEcell°, where n = mol e– transferred, F = Faraday constant, Ecell° = EC cell’s standard reduction potential
12.2. ATP
● Major energy carrier
○ Mid-level (ΔG°′ ≈ –30.5 kJ/mol): enough energy to power processes, while minimizing waste
○ Production: AMP/ADP/ATP
■ Substrate-level phosphorylation: directly phosphorylate ADP using coupled rxn.
■ Oxidative phosphorylation (ox. phos.): power ATP synthase using PMF from ETC
○ Stores energy in phosphate bonds: (–)-charged, mutual repulsion
○ Releases energy thru hydrolysis (breaking bonds is still endothermic)
● Coupling: fuels energetically unfavorable rxns. w/ ATP hydrolysis
● Phosphoryl group transfer: in/activate a molecule by transferring high-energy phosphate
12.3. Redox
● Half-rxns.: split redox rxns. into reduction (gain e–), oxidation (lose e–) components
● Spontaneous redox: ΔG < 0 (free energy), E > 0 (electromotive force)
● High-energy e– carriers
○ NADH, NADPH, FADH2, FMNH2, QH2, cyt cred, glutathione, etc.
○ Flavoproteins: contain riboflavin (vit. B2) cofactor
■ Flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN)
■ e– carriers in mitochondria/chloroplasts
■ Activate B vitamins
■ Coenzymes for β-oxidation, PDH, glutathione reduction
12.4. Metabolic States
● Homeostasis: maintain a physiologically stable state using energy
○ Not equilibrium! Equilibrium = death
● Postprandial/absorptive/well-fed state: abundant nutrients, lasts 3–5 hrs after meal
○ Anabolism, fuel storage > catabolism
○ Nutrients move from gut to liver (thru hepatic portal vein) to tissue
○ Blood sugar ↑ → insulin ↑
■ Tissues: glucose uptake
■ Liver: glycogenesis, then FA synthesis
● In this state, liver subsist mostly on excess AA metabolism
■ Fat: triacylglycerol synthesis
■ Muscle: protein synthesis
■ Nervous tissue, RBCs unaffected
● Nervous tissue subsists solely on aerobic respiration (or ketone bodies in prolonged fasting state)
● RBCs subsist solely on anaerobic glycolysis
● Postabsorptive/fasting state
○ Counterregulatory hormones (glucagon, cortisol, Epi, NE, growth hormone): oppose insulin
 ■ Liver: releases glucose, glycogenolysis (and some gluconeogenesis)
■ Fat: releases FAs (for acetyl-CoA)
■ Skeletal muscle: releases AAs (for gluconeogenesis)
● Prolonged fasting state/starvation
○ Use ketone bodies, not glucose, as major energy source
■ Avoid metabolizing AAs for gluconeogenesis
○ High glucagon, Epi
■ Liver: rapid glycogenolysis
■ Fat: rapid lipolysis (for ketone bodies)
■ Muscle: switches to burning FAs, slow AA breakdown (for gluconeogenesis for brain, RBCs)
■ Brain: switches to using ketone bodies (and some glucose)
■ RBCs must still use glucose
12.5. Hormonal Regulation of Metabolism
● Systemic regulation: thru covalent mechanisms (de/phosphorylation)
○ Peptides: rapid adjustments thru 2nd-messenger cascades (e.g., insulin)
○ AAs, steroids: long-range adjustments thru transcriptional control (e.g., thyroid hormones, cortisol)
● Insulin: well-fed, Glc uptake ↑, carb metabolism ↑, storage ↑
○ Peptide, secreted by pancreatic β cells (in Langerhans islets)
○ Controlled mainly by blood sugar
■ Glc metabolism → [ATP] ↑ in β cells → Ca 2+ release → insulin exocytosis
● Insulin secretion ∝ [blood sugar] when [blood sugar] > 100 mg/dL (~5.6 mM)
■ Also affected by glucagon, somatostatin
○ Need insulin for effective Glc uptake: resting skeletal muscle, fat
■ Store glucose if [Glc] is high
○ Don’t need insulin: nervous tissue, kidney tubules, intestinal mucosa, RBCs, pancreatic β cells
■ Absorb Glc even if [Glc] is low
○ Increases…
■ Glc uptake, carb metabolism in muscle, fat
■ Glycogenesis in liver, muscle (glucokinase, glycogen synthase ↑)
■ Glc, triacylglycerol uptake in fat
■ LPL activity (clears chylomicrons/VLDLs from blood)
■ Lipogenesis in liver, fat
○ Decreases…
■ Glycogenolysis in liver, muscle (glycogen phosphorylase, G6Pase ↓)
■ Lipolysis in fat
■ Ketone body synthesis in liver
● Glucagon: fasting, gluconeogenesis ↑, ketogenesis/FA metabolism ↑, storage ↓
○ Peptide, secreted by pancreatic α cells (in Langerhans islets)
 ○ Increases…
■ Glycogenolysis in liver (glycogen phosphorylase ↑, glycogen synthase ↓)
■ Gluconeogenesis in liver (PC, PEPCK, F1,6BPase ↑)
■ Ketogenesis in liver
■ Lipolysis in liver (HSL ↑)
○ Decreases…
■ Lipogenesis in liver
● Glucocorticoids: “fight or flight,” blood sugar ↑, Glc uptake ↓, storage ↓
○ Steroids, secreted by adr. cortex in stress (e.g., cortisol)
○ Increases…
■ Gluconeogenesis, AA breakdown in liver
■ Lipolysis in fat
■ Glucagon, Epi, other catecholamines
○ Decreases…
■ Glc uptake in muscle, fat, lymphoid tissue (reserves Glc for nervous tissue)
○ Long term: hyperglycemia → insulin ↑, fat storage ↑
● Catecholamines: “adrenaline rush”
○ AAs, secreted by adr. medulla (e.g., Epi/adrenaline, NE/noradrenaline)
○ Increases…
■ Glycogenolysis in liver, muscle (glycogen phosphorylase ↑)
● In skeletal muscles, resulting G6P is metabolized, not released into bloodstream (no G6Pase)
■ Lipolysis in fat (HSL ↑)
■ Basal metabolic rate (BMR) thru sympathetic responses
● Thyroid hormones: permissive (more or less constant)
○ AAs, secreted by thyroid (e.g., thyroxine/T4, triiodothyronine/T3)
■ T4 ⤚(deiodinases)→ T3 + I–
○ Increases…
■ BMR → oxygen consumption, heat production ↑
● T3 is faster-, shorter-acting than T4
■ Glc absorption in small intestine
■ Cholesterol clearance in blood
12.6. Tissue-Specific Metabolism
● Liver: maintains constant blood sugar, synthesizes ketone bodies in excess β-oxidation
○ Well-fed: subsists on excess AAs
■ Glycogenesis, FA synthesis (released as VLDLs)
○ Fasting: subsists on FAs
■ Glycogenolysis, gluconeogenesis (from lactate, glycerol, AAs)
● Fat: stores triacylglycerols
 ○ Well-fed: subsists on Glc
■ Release FAs from chylomicrons/VLDLs (LPL ↑), triacylglycerol synthesis
○ Fasting: subsists on FAs
■ Release FAs (HSL ↑)
● Skeletal muscle: major fuel consumer
○ Resting
■ Well-fed: subsists on Glc, FAs
● Glc uptake, glycogenesis, protein synthesis
■ Fasting: subsists on free FAs
■ Prolonged fasting: subsists on ketone bodies
○ Active
■ Very short term (2–7 s): subsists on creatine phosphate
■ Short term: subsists on anaerobic glycolysis of glycogen stores, blood sugar, free FAs
■ Long term (1–3 hrs): glycogen depleted, subsists on FAs
● Cardiac muscle
○ Well-fed, fasting: subsists on FAs
○ Prolonged fasting: subsists on ketone bodies
○ Failing heart: subsists more on Glc (anaerobic)
● Brain
○ Well-fed, fasting: needs continuous supply of Glc
■ FAs cannot cross blood–brain barrier
■ Hypoglycemia (< 70 mg/dL) → glucagon, Epi ↑
○ Prolonged fasting: subsists on ⅔ ketone bodies, ⅓ Glc
12.7. Integrative Metabolism
● Measuring metabolism
○ Measure blood sugar, thyroid hormones/TSH, insulin/glucagon, oxygen/CO2
○ Respirometry: measures respiratory quotient (RQ)
■ RQ = (CO2 produced)/(O2 consumed) for complete combustion of a fuel
● RQ ≈ 1 (carbs), 0.7 (lipids)
■ Resting RQ ≈ 0.8, changes depending on stress, starvation, exercise
○ Calorimetry: measures basal metabolic rate (BMR, amt. energy required for 1 sedentary day)
■ Measures heat exchange using large insulated chamber w/ heat sinks
■ Estimate w/ previous data using age, weight, height, gender
● Body mass regulation
○ Water: frequent, minor fluctuations in mass
○ Lipids: most responsible for gradual change in mass
○ Body mass ↑ → BMR ↑
■ Small changes in intake → offset w/ small changes in energy expenditure
 ■ Small changes in activity → offset w/ small changes in hunger
○ Ghrelin (hunger ↑), orexin (appetite ↑), leptin (orexin ↓ → appetite ↓)
○ Body mass index: BMI = (mass in kg)/(height in m)2
■ Underweight: < 18.5
■ Normal: 18.5–25
■ Overweight: 25–30
■ Obese: > 30