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MCAT Biochem
MCAT Biochem
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
2. Enzymes
2.1. Biological Catalysis
● Catalysts
○ Change kinetics: Ea ↓, easier to form/more stable 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 consumed in rxn.
○ Sensitive to pH, temp
● Major classifications: “LIL HOT”
○ Ligases: 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
○ Lyases: 1 molecule ⇌ 2 molecules
■ No hydrolysis, no redox
■ e.g., synthases
○ Hydrolases: hydrolysis
■ e.g., phosphatase, lipases, peptidases, nucleases
○ Oxidoreductases: redox
■ Has e–-carrier cofactors, like NAD+
■ Reductants (e– donors), oxidants (e– acceptors)
■ e.g., dehydrogenases, reductases, oxidases
○ Transferases: move functional groups between molecules
■ Kinases: transfer Pi
2.2. Mechanisms
● Provide favorable microenvironments (pH, charge), stabilize TS‡, bring reactive groups closer together
● Enzyme–substrate binding
○ Substrate binds to active site, forms enzyme–substrate complex
■ 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: 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.
● Michaelis–Menten (MM) kinetics
○ MM equation: E + S ⥫(k1/k–1)⥬ ES ⤚(kcat)→ E + 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
■ Low kcat: low turnover, slower rxn.
■ High kcat: high turnover, faster rxn.
■ Catalytic efficiency = kcat / Km
○ 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: (–1/Km, 0), (0, 1/vmax), slope = Km /vmax
○ Used to calculate Km, vmax
● Cooperativity
○ v vs. [S] graph is sigmoidal
○ 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 ↓
2.5. 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
■ 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
■ 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: activate/deactivate enzyme
■ Glycosylation: tag enzyme for transport, modify activity/selectivity
○ Zymogens: regulatory domain must be cleaved/modified to activate enzyme
■ e.g., dangerous enzymes like trypsin (digestion), caspases (apoptosis)
3.2. Biosignaling
● Ion channels: specific pathways for facilitated diffusion of ions
○ Ungated: unregulated
■ 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
○ 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
■ 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
4. Carbohydrates
4.1. Classification
● General formula: Cn(H2O)m
● Monosaccharides
○ Trioses (3 C’s), tetroses (4 C’s), pentoses (5 C’s), hexoses (6 C’s), etc.
○ Aldoses: aldehyde is most oxidized group
■ Simplest aldose: glyceraldehyde, CHO–(H)C(OH)–CH2OH
○ Ketoses: ketone is most oxidized group
■ Simplest ketose: dihydroxyacetone, CH2OH–CO–CH2OH
○ Common sugars: fructose, glucose, galactose, mannose
● Stereochem
○ Optical/stereoisomers: same connectivities, diff. spatial arrangement of atoms
■ Enantiomers: mirror images
● Chiral C: C w/ 4 diff. groups
● # stereoisomers = 2(# chiral C’s in molecule)
■ Diastereomers: not mirror images
● Epimers: differ at 1 chiral center only
○ Absolute configuration
■ (R)/(S): CIP priority rules, (R) is CW, (S) is CCW
■ (+)/(–): experimentally determined
■ ᴅ/ʟ: hydroxyl of penultimate C in Fischer projection (highest-number chiral C), ᴅ is right, ʟ is left
○ Fischer projection
■ Horizontal lines are out from page, vertical lines are into page
■ Highest-oxidized group is nearest to the top, start counting from that topmost C (C-1)
4.3. Monosaccharides
● Redox
○ Reducing sugars: hemiacetal cyclic sugars
■ Aldonic acid: open-chain aldose after oxidation (aldehyde → CA)
■ Lactone: cyclic aldose after oxidation (hemiacetal → ester) (anomeric C hydroxyl → carbonyl)
■ Alditol: aldose after reduction (aldehyde → alcohol)
■ 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) → Ag 2O (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
○ Phosphorylation: phosphate esters
● Glycosides
○ Hemiacetals/ketals + alcohols + cat. acid → acetals/ketals (glycosides)
■ Furanosides: glycosides from furanoses
■ Pyranosides: glycosides from pyranoses
○ Glycosidic bonds: [sugar]–OR
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–CH2–CH2–NMe3+)
■ Phosphatidylethanolamine: ethanolamine head group (⦚–O–CH2–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
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
6.3. 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), Mg 2+, 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), Mg 2+, 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 + PP i
● 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 Ca 2+-binding sites
○ Glycosylation: adds oligosaccharides, indicates cellular destination
○ Prenylation: adds lipid groups to membrane-bound enzymes
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
9.2. Glycolysis
● Breaks down glucose into 2 pyruvate, producing some energy
● Occurs in cytoplasm
● Net: glucose + NAD+ + 2 ADP + 4 P i → 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)
■ 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 + CO2
○ 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
9.5. 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 + PP i
● 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
○ 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 + P i
■ 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
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