Celbiologie I (2022-2023)

Praktisch

• 11stp (oud curriculum)
• eerste semester
• docenten
  • prof. A. Van Eynde
  • prof. M. Bollen
  • prof. R. Derua
• handboek: Becker’s World of the Cell (9th)
• onderdelen
  • 40 lessen (60u)
  • 6 werkzittingen (9u)
  • 5 practica (20u)
    • verplicht
    • labojas meebrengen
    • 10% punten
  • 3 sessies monitoraat (4.5u)
• examen
  • 90% punten
  • 3u
  • multiple choice
    • 60 vragen?
  • geen rekentoestel
    • dus ook geen formules uitwerken
    • wel formules herkennen
• https://drive.google.com/drive/folders/1OrHen5XHsQ2YURugr6Fx5F_mZAH2OKId

Formularium

• tabel met codon-AZ mapping wordt gegeven indien nodig
• $pH = pK_a + \log \dfrac{[\ce{A-}]}{[\ce{HA}]}$
• $G = \Delta H - T\Delta S$
• $\Delta G^{0\prime} = -RT \ln K'_{eq} = -592 \ln K'_{eq}$
• $\Delta G' = \Delta G^{0\prime} + RT \ln \dfrac{[C]^c[D]^d}{[A]^a[B]^b}$
• $v = \dfrac{V_{max} [S]}{K_m + [S]}$
• $V_{max} = k_{cat} [E_{tot}]$
• Lineweaver-Burk plot
  • $\dfrac{1}{v} = \dfrac{K_m}{V_{max}} \dfrac{1}{[S]} + \dfrac{1}{V_{max}}$
• Eadie-Hofstee plot
  • $\dfrac{v}{[S]} = \dfrac{V_{max}}{K_m} - \dfrac{1}{K_m}v$
• Hanes-Woolf
  • $\dfrac{[S]}{v} = \dfrac{K_m}{V_{max}} + \dfrac{1}{V_{max}}[S]$
• gasdichtheid bij STP: 22.4 L/mol
• $T = 298K$
• $R = \pu{1.987 cal//mol K}$
• $RT = \pu{592 cal//mol}$
• $F = \pu{23062 cal//mol V}$
• $\Delta G^{0\prime} = -nF\Delta E'_0 = -nF(pmf)$
• G(ATP): -7.3

Basics of organic chemistry

• TODO methyl, acetyl, acetone, formaldehyde, ...
• functional groups (at neutral pH)
  • $\oplus$
    • amino: $\ce{-NH3+}$
  • $\ominus$
    • carboxyl: $\ce{-(C=O)-O-}$
    • phosphate: $\ce{PO4^2-}$
  • neutral but polar (due to presence of $\ce{O}$ and $\ce{S}$)
    • hydroxyl: $\ce{-OH}$
    • sulfhydryl: $\ce{-SH}$
    • carbonyl: $\ce{-(C=O) -}$
      • in aldehydes, ketones, carboxylic acids, amides, ...
    • aldehyde: $\ce{-(C=O)-H}$
  • unclassified
    • acyl: $\ce{-CO -}$
      • oxoacid that lost 1 or more $\ce{-OH}$ groups
• classes
  • ester: $\ce{R-(C=O)-O-R'}$
  • ether: $\ce{R-O-R'}$
  • thio-ether: $\ce{R-S-R'}$
  • aldehyde: $\ce{R-(C=O)-H}$
  • ketone: $\ce{R-(C=O)-R'}$
  • amine
    • $\ce{RNH2}$
    • $\ce{R2NH}$
    • $\ce{R3N}$
    • $\ce{R4N+}$
  • thiol: $\ce{R-SH}$
  • thial: $\ce{R-(C=S)-H}$
  • phosphate: $\ce{R-H2PO4}$
  • oxoacid: acid that contains $\ce{O}$
    • carboxylic acid: $\ce{R-(C=O)-OH}$ = $\ce{R-COOH}$
      • deprotonation: $\ce{R-COO-}$
• acid-base
  • $pX = -\log [\ce{X}]$
  • $pH = -\log [\ce{H+}]$
  • acid: $\ce{HA <=> H+ + A-}$
    • $\ce{HA}$: acid
    • $\ce{A-}$: conjugated base
    • $K_a = \dfrac{[\ce{H+}] [\ce{A-}]}{[\ce{HA}]}$
    • $\iff [\ce{H+}] = K_a \dfrac{[\ce{HA}]}{[\ce{A-}]}$
    • $\iff pH = pK_a - \log \dfrac{[\ce{HA}]}{[\ce{A-}]}$
    • $\iff pH = pK_a + \log \dfrac{[\ce{A-}]}{[\ce{HA}]}$
      • Henderson–Hasselbalch equation
  • base: $\ce{H2O + B <=> BH+ + OH-}$
    • $K_b = \dfrac{[\ce{BH+}][\ce{OH-}]}{[\ce{B}]}$
    • $\iff [\ce{OH-}] = K_b\dfrac{[\ce{B}]}{[\ce{BH+}]}$
    • $\iff pOH = pK_b - \log \dfrac{[\ce{B}]}{[\ce{BH+}]}$
    • $\iff pOH = pK_b + \log \dfrac{[\ce{BH+}]}{[\ce{B}]}$
• redox
  • oxidation
    • donate electrons
    • release energy
    • e.g., $\ce{C6H12O6 + 6O2 -> 6CO2 + 6H2O}$
  • reduction
    • accept electrons
    • requires energy
    • e.g., photosynthesis

H1 Preview

• cell theory
  • all organisms consist of 1 or more cells
  • cell is basic unit of structure for all organisms
  • all cells arise only from preexisting cells

Strand 1: cytology

• sizes
  • small molecules: <1nm
  • DNA: 2nm
  • microfilament: 7nm
  • membrane: 8nm
  • microtubule: 25nm
  • ribosome: 25nm
  • virus: 100nm
  • mitochondria: 1um
  • bacteria
  • nucleus: 10um
  • eukaryotic cell: 10-100um
• microscopy
  • microtome: cut in small slices
  • dyes and stains
  • resolution (length): how far apart to see as 2 entities?
    • $0.5\lambda \in [200, 350]$ nm
    • eye: 0.25mm
    • light microscope: 0.25um (1000x)
    • electron microscope: 2nm (100 000x)
  • types of light microscopy
    • brightfield
      • requires fixation and (usually) staining -> dead cells
    • phase contrast
      • alive
      • based on subtle phase change of light during refraction
    • differential interference contrast
      • idem
    • fluorescence
      • antibody labeling = immunofluorescence
        • binds to specific antigen
        • primary=direct: C region of antibody is labeled
        • secondary=indirect
          • primary antibody attaches to antigen
          • labeled secondary antibody attaches to primary antibody
          • can bind multiple times -> amplify signal -> more sensitive
      • green fluorescent protein (GFP) from jellyfish
    • confocal
      • focus on 1 plane using LASER
  • electron microscopy
    • shorter wavelength -> better resolution
    • 2 types
      • transmission (TEM): through tissue (2D)
      • scanning (SEM): 3D effect, only surface

Strand 2: biochemistry

• methods
  • isotopes (e.g., C14)
  • centrifugation = subcellular fractionation
  • chromatography (general term)
    • separate mixture of molecules based on charge, size or affinity
    • added to system (tube, paper, ...) with stationary phase material
    • techniques
      • thin layer chromatography (TLC)
        • stationary phase: cellulose or silica gel layer
        • components travel through layer at different rates
        • application/namesake: separating plant pigments
      • column chromatography
        • stationary phase in tube
        • then add mixture
        • components will diffuse/sink at different rates
  • electrophoresis
    • electric field
    • separate macromolecules based on mobility (~size, charge) through gel
    • useful for DNA, RNA, proteins
  • mass spectrometry
    • determine size (mass?) and composition of proteins

Strand 3: genetics

• chromosome theory of heredity
• Watson & Crick: DNA double helix
• central dogma of molecular biology: DNA -> RNA -> proteins
  • replication
  • transcription
  • translation
• methods
  • recombinant DNA tech
    • = DNA from different sources combined
    • using restriction enzymes: cleave DNA
    • hybridization: make double strand out of two single strands
  • sequencing
  • bio-informatics
• model organisms
  • bacterium: Escherichia coli
    • 4000 genes
    • unicellular, prokaryote
    • macromolecule synthesis
  • yeast: Saccharomyces cerevisiae
    • 6000 genes
    • unicellular, eukaryote
    • cell cycle
  • fruit fly: Drosophila melanogaster
    • 13 000 genes
    • developmental biology
  • roundworm: Caenorhabditis elegans
    • 19 000 genes
    • developmental biology, aging
  • mouse: Mus musculus
    • 25 000 genes
    • mammal
    • human pathologies
  • green alga: Chlamydomonas reinhardtie
    • unicellular, eukaryote, plant
  • Arabidopsis thaliana
    • 25 000 genes
    • multicellular, flowering plant
    • "zandraket"

H2 Chemistry of the cell

• calorie (cal):
  • energy needed to raise the temp of 1g water with 1K
  • $\pu{1J = 0.239 cal}$
  • $\pu{1 cal = 4,184 J}$
  • $\pu{1 kcal = 4 184 J}$
• bond strength (strong->weak)
  • covalent
    • $\ce{C#C}$
    • $\ce{C=C}$
    • $\ce{C-H}$
    • $\ce{C-O}$
    • $\ce{C-C}$
    • $\ce{C-N}$
  • visible light photon energy
  • hydrogen bond (1/10 strength)
  • thermal vibration energy
• asymmetric $\ce{C}$ atom: four different groups
  • $n$ asymmetric atoms give rise to $2^n$ stereoisomers
  • examples
    • one per amino acid
      • D-alanine
        • amino group right in Fischer
      • L-alanine
        • amino group left in Fischer
      • both occur in biology, only L-alanine used in proteins
    • four in (linear) aldohexose (see H3)
• cell composition
  • 70% water
  • 30% chemicals
    • 15% proteins
    • 6% RNA
    • 4% ions and small molecules
    • 2% phospholipids
    • 2% polysaccharides
    • 1% DNA
• properties of water
  • polar
  • cohesive due to hydrogen bonds
  • high specific heat and high heat of vaporization due to hydrogen bonds
  • excellent solvent
    • hydrophilic, hydrophobic or both (amphipathic)
  • $pH = pK_a + \log \dfrac{[\ce{A-}]}{[\ce{HA}]}$
• buffers
  • intracellular: $\ce{H2PO4-}$ en $\ce{HPO4^2-}$, $pK=7.2$
  • extracellular: $\ce{H2CO3}$ en $\ce{HCO3-}$, $pK=6.1$
    • $\ce{H2CO3 <=> CO2 + H2O}$
• membranes
  • phospholipids and glycolipids form lipid bilayer
    • polar head: hydrophilic
    • nonpolar tail: hydrophobic
  • permeability (good -> poor)
    • nonpolar
    • small, uncharged, polar
    • large, uncharged, polar
    • charged (ions)
• macromolecules
  • proteins, nucleic acids, polysaccharides, lipids
  • monomer -> polymer
    • $\ce{H-X-OH + H-X-OH -> H-X-X-OH + H2O}$
    • amino acids -> proteins
    • nucleotides -> nucleic acids
    • monosaccharides -> polysaccharides
    • (lipids are not polymers)
  • synthesis: condensation = dehydration (requires energy)
  • analysis: hydrolysis (releases energy)
  • 3D structure
    • self-assembly
      • might require chaperones
    • hydrophobic hydration
      • H bonds
        • donor vs acceptor?
      • ionic interactions
      • hydrophobic interactions
    • Van Der Waals interactions
    • depends on T, pH

H3 Macromolecules

Proteins

• nine classes
  • enzymes
  • structural
  • motility
  • regulatory
  • transport
  • signaling
  • receptor
  • defensive
  • storage
• monomer: amino acid (AA)
  • 
  • 20 types used in proteins
    • 60+ exist (incl. posttranslational modifications)
  • three letter code is usually first three letters of name
    • exceptions: Asn (vs Asp), Gln (vs Glu), Ile, Trp
  • chiral $\ce{C_\alpha}$
    • four covalent bonds
      • amino group: $\ce{NH3+}$
        • background info
          • primary amine $\ce{R-NH2}$, or $\ce{R-NH3+}$ protonated
          • secondary amine $\ce{R-NH-R'}$, or $\ce{R-NH2+-R'}$ protonated
        • ionized (protonated) at physiological pH
      • carboxyl group: $\ce{COO-}$
        • ionized (deprotonated) at physiological pH
      • $\ce{H}$ atom
      • $\ce{R}$ group
        • classification
          • 9 nonpolar, hydrophobic, found on inside or in membrane
            • Gly, Ala,Val, Leu, Ile, Met, Phe, Trp, Pro
            • mostly $\ce{C}$ and $\ce{H}$
            • Met contains $\ce{S}$
            • Trp contains $\ce{N}$
            • Phe and Trp contain aromatic group
            • Pro contains secondary ($\ce{NH2+}$) instead of primary amine
              • due to ring
              • secondary amino acid (formerly imino acid)
          • 11 polar, hydrophilic, found on outside
            • 6 uncharged
              • due to presence of $\ce{O}$ or $\ce{S}$ (or $\ce{N}$?)
              • Ser, Thr, Cys, Tyr, Asn, Gln
              • Cys contains $\ce{S}$
              • Asn, Gln contain $\ce{NH2}$
              • Tyr contains aromatic group
            • 5 charged
              • 2 acidic, negative
                • Asp, Glu
                • due to $\ce{COO-}$
              • 3 basic, positive
                • Lys: $\ce{NH3+}$
                • Arg: $\ce{NH2+}$
                • His: $\ce{NH+}$
                  • can be acidic of basic
                    • pK=6 when free in water
                    • at pH=7.4
                      • 4% $\ce{NH+}$: acidic, pH=5.8
                      • 96% $\ce{N}$: basic, pH=7.8
                      • both exists!
                      • application
                        • acid-base catalysis in RNase
                    • pK can shift due to surroundings
                      • hydrophobic env -> less acidic
                      • basic env -> more acidic
                        • stabilize negative charges???
                    • still good buffer
    • two stereoisomers: L (in proteins) and D
      • except Glycine (because $\ce{R} = \ce{H}$)
• polymer: polypeptides and proteins
  • peptide bond (covalent)
    • amino acids -> polypeptide
    • $\ce{R-COO- + NH3+-R' -> R-CO-NH-R' + H2O}$
    • six atoms between $\ce{C_\alpha}$ are planar due to partial double bond
      • i.e., fixed $\ce{C-N}$ connecting two AA
      • exception: can also rotate in Pro
    • $\ce{N}$ terminus and $\ce{C}$ terminus
      • created from N -> C during translation
    • amino acids become amino acid residues
    • cis vs trans
      • by default trans: $\ce{C_\alpha}$ on different sides
      • exception: Pro can be cis
  • protein: one or more polypeptides with unique, stable 3D structure, active
    • monomeric: single polypeptide
    • multimeric: multiple polypeptides
      • dimer: e.g., insulin
      • trimer
      • tetramer: e.g., hemoglobin ($\alpha_2\beta_2$)
• folding
  • into proper shape = conformation
  • denaturation and renaturation
    • depends on temperature, pH, salt, ...
  • caused by bonds and interactions
    • between $\ce{R}$ groups of amino acids
    • covalent (70-100kcal/mol, 150-200kcal/mol for double/triple bonds)
      • anisotropic (why?)
      • disulfide bond
        • between two Cys that each contain sulfhydryl groups
        • $\ce{R-SH + SH-R' -> R-S-S-R' + 2H}$
        • oxidation
        • intra- vs intermolecular
    • non-covalent
      • hydrogen bonds (5kcal/mol)
        • weak but abundant
        • important for alpha helices and beta sheets
        • donor: $\ce{H}$ with $\delta^+$ in e.g., $\ce{OH}$ and amino groups
          • also those not in R?
        • acceptor: $\delta^-$ atom in e.g., $\ce{C=O}$ and $\ce{SH}$
      • ionic bonds (3kcal/mol)
        • long distance, isotropic force
        • depends on pH to maintain ionization
      • Van Der Waals interactions (0.1-0.2kcal/mol)
        • transient dipoles
        • close range: < 0.2nm
        • important in context of complementary surfaces
      • hydrophobic interactions
        • hydrophobic inside
        • hydrophilic outside
        • combination allows compact 3D shape
• levels
  • primary structure
    • amino acid sequence
    • covalent peptide bonds
    • N -> C
  • secondary
    • Ramachandran diagram
      • rotation angles between $\ce{C_\alpha}$ and resp. N and C
    • H bonds in backbone (not sidechains) between $\ce{NH}$ and $\ce{CO}$
    • alpha helix
      • right handed
      • intramolecular
        • $AA_i \to AA_{i+3}$
      • 3.6 AA/turn
      • $\ce{R}$ point outward (steric hindrance)
      • typical AAs
        • Leu, Met: non-polar
        • Glu: acidic
      • helix breaker
        • Pro: side chain interferes with backbone
        • Gly: too flexible around $\ce{C_\alpha}$
      • hydrphobic helices in membranes
      • amphipathic helices
        • repeats of 2 hydrophobic and 2 hydrophilic AA
    • beta sheet
      • intra or intermolecular
      • parallel or antiparallel
      • Ile, Val, Phe: non-polar
      • type
        • hydrophilic
        • hydrophobic
        • amphipathic
          • on separate sides of sheet
    • motifs
      • beta-alpha-beta
      • hairpin loop (beta-turn-beta)
      • helix-turn-helix
  • tertiary
    • based on R-group interactions
    • stabilized by disulfide bonds
    • also non-covalent bonds
    • two types
      • fibrous
        • secondary > tertiary
        • fibroin in silk
          • small R groups: Gly, Ala, Ser
          • lots of beta sheets
            • already max stretched
            • creases/wrinkles possible
        • keratin in hair
          • lots of alpha helices
            • extensible
          • coiled coil
            • hydrophobic R -> tight packing
        • collagen
          • Gly, Pro -> left handed helix
          • 3x -> right handed helix
        • elastin
      • globular
        • most proteins
        • ribonuclease
        • domains
          • locally folded structure
          • separate function
  • quaternary
    • multiple polypeptides -> multimeric protein
      • "subunits" of "chains"
    • bonds: = tertiary
  • multiprotein complex ("pipeline")
    • pyruvate dehydrogenase complex
• insulin
  • subunit A (21AZ)
    • 1 intramolecural disulfide bond
  • subunit B (30AZ)
  • 2 intermolecular disulfide bonds
• hemoglobin
  • two alpha subunits
  • two beta subunits
  • sickle cell
    • beta subunit: glutamate -> valine (E6V)
    • deoxyHb sticks together
      • zie werkzitting 1, oef 16
• Alzheimer
  • amyloid plaques
    • outside neurons, near synapses
  • tau polymer
    • inside neurons
    • neurofibrillary knots
• X-ray cristallography
  • prerequisite: primary structure known
  • create pure protein crystal
    • decrease solubility
      • add precipitans (salt?) -> less water
      • supersaturated -> crystal
  • irradiation of crystal at various angles
    • result: diffraction pattern
  • model construction

Nucleic acids

• heterocyclic aromatic bases
  • pyrimidines (1 6-ring)
    • cytosine (C)
    • thymine (T) (DNA only)
    • uracil (U) (RNA only)
  • purine (6 ring [pyrimidine] + 5 ring [imidazol])
    • adenine (A)
    • guanine (G)
  • somewhat hydrophobic
  • alternating double bonds -> resonance -> very stable
• sugar: aldopentose
  • 1' - 5'
  • D-ribose
  • D-deoxyribose ($\ce{H}$ instead of $\ce{OH}$ at 2')
• nucleoside = base + sugar at 1'
  • (deoxy)adenosine
  • (deoxy)guanosine
  • (deoxy)cytidine
  • deoxythymidine
  • uridine
• nucleotide = nucleotide + $\ce{PO4^2-}$ at 5'
  • AMP / dAMP
  • GMP / dGMP
  • CMP / dCMP
  • • / dTMP
  • UMP / -
• nucleotide -> polynucleotide = nucleic acid
  • phosphodiester bridge
  • 5' -> 3'
    • terminal 5' has P
    • terminal 3' has -OH end
  • sugar-phosphate backbone
    • charged P
    • polar $\ce{OH}$s in ribose
    • hydrophilic
• synthesis requires energy and info
  • DNA: dATP, dCTP, dGTP, dTTP
  • RNA: ATP, CTP, GTP, UTP
• phosphorylated adenosine
  • AMP
    • 1 phosphoester bond
    • A-P
  • ADP
    • 1 phosphoester bond
    • 1 phosphoanhydride bond
    • A-P~P
  • ATP
    • 1 phosphoester bond
    • 2 phosphoanhydride bonds
    • A-P~P~P
• DNA
  • 
  • right-handed double stranded (ds) helix (for B-DNA)
  • antiparallel
    • 5'->3': coding strand
    • 3'->5': template strand = matrijs, from which RNA is transcribed
    • roles can be flipped by "rotating DNA"
  • complementary base pairing
    • purine + pyrimidine (to keep width equal)
      • $\ce{A=T}$
      • $\ce{G#C}$
  • base stacking
    • hydrophobic interactions
    • stabalizes structure
    • G-C more than A-T?
  • major and minor groove
• RNA
  • mostly (not always) single stranded (ss)
  • ribose less stable in basic environment than deoxyribose
    • due to $\ce{OH}$ group
  • more flexbile -> more functions than DNA
  • secondary structure: bind with self
    • hairpin
    • stem-loop: $\Omega$

Polysaccharides

• background: hemi-acetal and hemi-ketal
  • aldehyde: $\ce{R-(C=O)-H}$
  • keton: $\ce{R-(C=O)-R'}$
    • e.g. aceton = dimethylketon $\ce{CH3-(C=O)-CH3}$
  • alcohol: $\ce{R2-OH}$
  • aldehyde + alcohol -> hemi-acetal
    • C with 4 groups
      • $\ce{H, OH, R, OR2}$
    • can reduce atoms by oxidation of aldehyde
      • $\ce{R-(C=O)-H -> R-(C=O)-OH}$
      • $\ce{Cu^2+ -> Cu2O}$ (copper reduced from +II to +I)
  • ketone + alcohol -> hemi-ketal
    • C with 4 groups
      • $\ce{R', OH, R, OR2}$
    • cannot reduce
      • but ketose can transform into aldose in weak basic environment
      • via enadiol

Monosaccharides

• colloquial: "carbohydrates"
  • $\ce{C_nH_{2n}O_n} \approx \ce{C_n (H2O)_n}$
• aldose: terminal carbonyl group $\ce{C=O}$
• ketose: internal carbonyl group $\ce{C=O}$
  • typically at C2 -> 2-ketose
• $\ce{ketose <=>[heat] aldose}$
  • weak basic env helps
• 3-7 carbon atoms
  • numbering: start counting from most oxidized end (=at carbonyl group end)
  • triose
    • aldotriose
      • D-glyceraldehyde
      • L-glyceraldehyde
    • ketotriose
      • dihydroxyacetone (DHA)
    • note: pyruvate is not a sugar
  • tetrose
  • pentose
    • aldopentose
      • D-ribose
        • furanose 5-ring
          • 4C + O
          • 1 external C
  • hexose $\ce{C6H12O6}$
    • aldohexose
      • $2^{n-2} = 2^4 = 16$ stereoisomers
        • first and last carbon don't have 4 different groups
        • $\ce{C_{n-1}}$ determines D/L
          • D: $\ce{OH}$ right
          • L: $\ce{OH}$ left
        • $2^{n-3} = 8$ remaining per class
      • 8x D ("All altruists gladly make gum in gallon tanks")
        • D-allose
        • D-altrose
        • D-glucose
          • form
            • linear=straight chain -> Fischer projection
            • pyranose 6-ring -> Haworth projection
              • 5C + O
              • 1 external C
              • closed with hemi-acetal
                • aldehyde at C1
                • alcohol: $\ce{R2-OH}$ at C5
              • C1 now also chiral
                • $\alpha$-D-glucose: $\ce{OH}$ at C1 down
                  • starch, glycogen
                • $\beta$-D-glucose: $\ce{OH}$ at C1 up
                  • cellulose
              • conformations: chair vs boat
          • $\beta$-glucosamine
            • $\ce{OH -> NH2}$ at C2
        • D-mannose
        • D-gulose
        • D-idose
        • D-galactose
        • D-talose
      • 8x L (idem)
    • 2-ketohexose
      • $2^{n-3} = 2^3 = 8$ stereoisomers
      • 4x D
        • D-fructose
          • two ring forms
            • furanose 5-ring (most common)
              • 4C + O
              • 2 external C
              • closed with hemi-ketal
            • pyranose 6-ring
      • 4x L (idem)
  • heptose

Disaccharides

• covalent bonds
• condensation reaction
• glycosidic bond
  • $\alpha$ or $\beta$ depending on configuration of C1 in link
  • $\alpha$-glycosidic bond└┘
    • maltose $\alpha(1 \to 4)$ = $\alpha$-D-glucose + $\alpha$-D-glucose
      • reducing? yes, at C1 of left glucose
    • sucrose $\alpha(1 \to 2)\beta$ = $\alpha$-D-glucose + $\beta$-D-fructose
      • = saccharose
      • reducing? no, because TODO
  • $\beta$-glycosidic bond |‾|_|
    • lactose $\beta(1 \to 4)$ = $\beta$-D-galactose + $\beta$-D-glucose
      • lactose intolerance: lack enzyme to break this $\beta$ bond
      • reducing? yes, at C1 of left glucose

Polysaccharides

• storage
  • $\alpha$-D-glucose polymers
    • $\alpha(1 \to 4)$ bonds in backbone
      • cf. maltose
    • $\alpha(1 \to 6)$ bonds form sidechains
    • examples
      • starch (plants)
        • hydrophilic
        • amylose (unbranched)
          • hydrophobic
        • amylopectin (branched)
          • fewer longer branches
          • ~80% of starch
      • glycogen (animals and bacteria)
        • many short branches
        • hydrophilic
• structural
  • $\beta$-D-glucose polymers
    • $\beta(1 \to 4)$ bonds
      • mammals cannot hydrolyze this bond
    • examples
      • cellulose
        • in cell walls of plants
        • x36 -> microfibrils
          • rigid, lineair rods
        • hydrophobic
  • bacteria cell wall
    • alternating $\beta(1 \to 4)$ polysaccharide of
      • N-acetylglucosamine (GlcNAc)
      • N-acetylmuramic acid (MurNAc)
  • chitin in exoskeletons
    • GlcNAc only with $\beta(1 \to 4)$ bonds

Lipids

• hydrophobic or amphipathic
• not polymers in the classic sense
• six classes
  • fatty acids
    • building block
    • amphipathic
      • carboxyl group $\ce{COOH}$ = head = hydrophilic
      • hydrophobic tail
    • 12-20 carbons (even)
    • highly reduced (few oxygen atoms)
      • high energy potential upon oxidation
    • saturation
      • saturated: no double bonds, max number of $\ce{H}$
        • often animal fat
          • solid at room temp
      • unsaturated
        • one or more double bonds
        • kink
        • often vegetable oil
          • liquid at room temp
        • trans fats
  • triacylglycerols = triglycerides
    • glycerol + 3 fatty acids
      • glycerol: $\ce{CH2OH-CHOH-CH2OH}$
      • ester bonds
        • condensation
        • $\ce{R-OH + COOH-R' -> R-O-CO-R' + H2O}$
    • synthesis
      • monoacylglycerol
      • diacylglycerol
      • triacylglycerol
    • purpose
      • energy store
      • insulation
    • hydrophobic
  • phospholipids
    • amphipathic
      • more than fatty acids
    • in lipid bilayer membranes
    • two types
      • phosphoglycerides
        • composed of
          • glycerol
          • 2 fatty acids
            • 16-18 C
            • same ester bond as triglycerides
          • P
          • hydrophilic group $\ce{R}$
            • serine
            • ethanolamine
            • choline
            • inositol
      • sphingolipids
        • similar
        • glycerol -> sphingosine
        • only 1 fatty acid (up to 34 C)
          • amide bond
        • in lipid rafts
  • glycolipids
    • similar to phospholipids
      • P replaced by polysaccharide chain
  • steroids
    • hydrophobic
    • four ringed skeleton
    • variation in
      • functional groups
      • double bonds
    • only in eukaryotes
    • examples
      • cholesterol
        • amphipathic
      • estrogen: estradiol
      • androgen: testosterone
      • glucocorticoid: cortisol
      • mineralocorticoid: aldosterone
  • terpenes
  • = isoprenoids (made from isoprene)
  • vitamin A precursor
• solubility
  • triglycerides < fatty acids < phosphoglycerides

H5 Bioenergetics

• types of energy
  • synthetic
  • mechanical
  • concentration
  • electrical
  • heat
    • homeotherm (stable body temp)
    • poikilotherm (fluctuating)
  • light
    • bioluminescence
      • luciferine
    • not: fluorescence
    • phototrophy: light -> energy
      • photo-autotrophy: photosynthesis in plants
      • photo-heterotrophy: use light + organic molecules
    • chemotrophy: oxidation -> energy
      • chemo-autotrophy: anorganic molecules -> energy
      • chemo-heterothrophy: macromolecules -> energy
• temperature $T$
  • [T] = K (not Celcius!)
• internal energy $E$
  • [E] = kcal/mol
  • $\Delta E = E_{products} - E_{reagentia}$
• enthalpy/heat $H = E + PV$ - [H] = kcal/mol
  • in biology: constant pressure, temp and volume
  • $\Delta H = \Delta E$
    • $\Delta H < 0$: exotherm
    • $\Delta H > 0$: endotherm
• entropy $S$
  • [S] = kcal/K
  • $\dfrac{dS}{dt} \geq 0$
  • 2nd law thermodynamics: $\delta Q = TdS$ (background info)
  • spontaneity metric
  • $\Delta S_{universe} > 0$
    • open systems can have decreasing entropy if compensated elsewhere
  • increases with more disorder (TODO)
    • split (hydrolysis, ...)
    • better distribution (melting, evaporation, ...)
    • ...
• Gibbs free energy $G = \Delta H - T\Delta S$
  • [G] = kcal/mol
  • $\Delta G < 0$: spontaneous to right, exergonic = energy-yielding
  • $\Delta G > 0$: spontaneous to left, endergonic = energy-requiring
  • minimal $\iff K=K_{eq} \iff \Delta G = 0$
  • note: spontaneous != fast
• $\ce{aA + bB <=> cC + dD}$
  • interpretation $K_{eq}$
    • $> 1$: more products
    • $< 1$: more reagentia
  • $\Delta G = -RT \ln K_{eq} + RT \ln \dfrac{[C]^c[D]^d}{[A]^a[B]^b}$
  • $\Delta G^0$ -> standard conditions
    • $T = \pu{25^oC \approx 298 K}$
    • $RT = \pu{592 kcal/mol}$
    • $P = \pu{1 atm}$
    • $\forall X: [X] = \pu{1 M}$
  • $\Delta G^{0\prime}$
    • also pH=7
  • $\Delta G^{0\prime} = -RT \ln K'_{eq} = -592 \ln K'_{eq}$
    • $\Delta G^{0\prime} < 0 \iff K_{eq} > 1$
  • $\Delta G' = \Delta G^{0\prime} + RT \ln \dfrac{[C]^c[D]^d}{[A]^a[B]^b}$
• examples
  • hydrophobic hydratation
    • $\Delta S$ large (+)
      • because hydrophobic mix spontaneously with other hydrophobic elements
      • $-T\Delta S < 0$
    • $\Delta H$ small (+)
      • H bonds destroyed but also recreated
    • so $\Delta G < 0$
  • oxidation of glucose under standard conditions
    • $\ce{C6H12O6 + 6O2 -> 6CO2 + 6H2O + energy}$
    • $\Delta H = \pu{-673 kcal/mol}$: exotherm
    • $-T\Delta S = \pu{-13 kcal/mol}$
    • $\Delta G = \pu{-686 kcal/mol}$: spontaneous to right
  • photosynthesis under standard conditions
    • $\ce{6CO2 + 6H2O + energy -> C6H12O6 + 6O2}$
    • all signs reversed
    • $\Delta G = \pu{+686 kcal/mol}$: not spontaneous
  • $\ce{glucose-6-P <=> fructose-6-P}$
    • cf. $\ce{aldose <=> ketose}$
    • enzyme: phosphoglucoisomerase (PGI)
    • part of glycolysis
    • $K'_{eq} = 0.5$
      • twice as much glucose as fructose in equilibrium
      • $\Delta G^{0\prime} = -592\ln 0.5 = \pu{410 kcal/mol} > 0$
    • cells keep steady state away from equilibrium using ATP
      • [glucose-6-P] = $\pu{83 \mu M}$
      • [fructose-6-P] = $\pu{14 \mu M}$
      • $\Delta G' = \Delta G^{0\prime} + 592\ln\dfrac{14}{83} \approx \pu{-644 kcal/mol} < 0$

H5bis Bioenergetics

• druk $P$
  • [P] = Pa
• volume $V$
  • [V] = $m^3$ of L
• absolute temperatuur $T$
  • [T] = K
• hoeveelheid $n$
  • [n] = mol
• energie
  • in cal
    • energie vereist om 1g water met 1K te verhogen
  • 1 J = 0.239 cal
• gasconstante $R \approx 1.987$ cal/(K mol)
  • cal, niet kcal!
• moleculaire massa $M_r = 1$ Da $\iff M = 1$ g/mol (molaire massa, bij benadering)
• concentratiebreuk voor $\ce{aA + bB <=> cC + dD}$
  • $Q = \dfrac{[C]^c [D]^d}{[A]^a [B]^b}$
• evenwichtsconstante $K_{eq}$: waarde van $Q$ bij evenwicht
• interne energie $E$ (soms ook $U$)
  • [E] = cal/mol (of J/mol)
  • $\Delta E = E_{producten} - E_{reagentie}$
• enthalpie $H = E + PV \approx E$
  • [H] = cal/mol
  • $P$ en $V$ constant in biochemie
  • $\Delta H = \Delta E$
  • $\Delta H < 0$: exotherm
  • $\Delta H > 0$: endotherm
• entropie $S$
  • [S] = cal/(K mol)
  • cf. tweede hoofdwet thermodynamica: $\delta Q \geq TdS \iff dS \leq \dfrac{\delta Q}{T}$
  • $\Delta S_{universum} > 0$
  • $\dfrac{dS}{dt} \geq 0$
  • stijgt als
    • betere verdeling
    • splitsing in meerdere delen
    • ...
• Gibbs vrije energie $G$
  • [G] = cal/mol
  • $\Delta G = \Delta H - T \Delta S$
  • $\Delta G < 0$: exergonisch, reactie naar rechts spontaan
  • $\Delta G > 0$: endergonisch, reactie naar links spontaan
  • $G$ minimaal $\iff Q=K_{eq} \iff \Delta G=0$
  • $\Delta G^0$ onder "standard temperature and pressure" (STP)
    • T = 25 °C = 298.15 K
    • P = 1 atm
    • [P] = [R] = 1 M
    • gasdichtheid bij STP: 22.4 L/mol
    • onrealistisch in biochemie
  • $\Delta G^{0\prime}$
    • idem, plus pH = 7
  • voor reactie $\ce{aA + bB <=> cC + dD}$
    • $\Delta G = -RT \ln K_{eq} + RT \ln \dfrac{[C]^c [D]^d}{[A]^a [B]^b}$
    • $\Delta G^{0\prime} = -RT \ln K_{eq} = \pu{-592 cal//mol} \ln K_{eq}$
      • $\Delta G^{0\prime} < 0$ cal/mol $\iff K_{eq} > 1$
        • enkel voor $\Delta G^{0\prime}$
      • opgelet: in oef alles omzetten van kcal naar cal
    • $\Delta G' = \Delta G^{0\prime} + RT \ln \dfrac{[C]^c [D]^d}{[A]^a [B]^b}$
• voorbeelden
  • $\ce{C6H12O6 + 6O2 -> 6CO2 + 6H2O}$
    • $\Delta H = -673$ kcal/mol glucose
    • $T\Delta S = 13$ kcal/mol
    • $\Delta G = -673 - 13 = -686$ kcal/mol
  • hydrofobe hydratatie
    • $\Delta H$ stijgt beetje
    • $\Delta S$ stijgt veel
    • $\implies \Delta G = H - T\Delta S < 0$

H6 Enzymes

• enzymes
  • $\ce{substrate S ->[enzyme E] product P}$
  • reusable, stay same before and after reaction
    • $\ce{S + E <=> SE <=> PE <=> P + E}$
  • help speed up catalysis
    • bind all substrates to avoid relying on random collisions
    • lower activation energy $E_A$
      • conformation change -> "bijna transitietoestand"
    • surround substrates with strategic groups
• in context of enzymes
  • reagentia = substrate
  • binding site = active site
• enzyme-substrate interaction
  • $\ce{S + E <=> SE}$
  • dissociation constant $K_d = K_{eq}^{-1} = \dfrac{[S][E]}{[SE]}$
  • non-covalent
  • affinity
  • specificity
  • lock-key (old) vs induced fit (new)
• co-factors = prosthetic groups
  • metal ions
  • co-enzymes = small organic molecules (vitamin derivatives)
• enzyme = apo-enzyme + co-enzyme
• acid-base catalysis
  • protease: cleave proteins
  • base $\ce{NH+}$ attracts $\ce{e-}$ of $\ce{C=O}$ away from binding site
  • acid $\ce{COO-}$ repels $\ce{e-}$ of $\ce{H2O}$ towards binding site
  • cf. Gly-2 and Gly-5
• lysozyme: covalent catalysis
  • step 1: create covalent bond
  • step 2: hydrolysis
  • can cut bacterial cell wall (GlcNAc-MurNAc)
• sensitive to
  • pH: changes charges of amino acid side chains
  • ionic concentrations (e.g., salt)
  • feedback: inhibition or activation

Classification

• https://en.wikipedia.org/wiki/Enzyme_Commission_number
• EC 1 oxidoreductases
  • oxidation
    • e.g., ethanol -> acetaldehyde
    • $\ce{H-C-OH -> C=O + H+ + H-}$ (ignoring $\ce{R1, R2}$)
  • paired reduction
    • e.g., $\ce{NAD+ + H+ + H- -> NADH + H+}$
  • extract
    • proton $\ce{H+}$ from $\ce{C-OH}$
    • hydride ion $\ce{H-}$ from $\ce{C-H}$
• EC 2 transferases: $\ce{AB + C -> A + BC}$
  • often involve ATP
  • EC 2.7 ~phosphotransferases ($\ce{AP + B -> A + BP}$)
    • = dephosphorylation + phosphorylation?
    • EC 2.7.1 kinases
      • EC 2.7.1.1 hexokinase
        • $\ce{glucose + ATP -> glucose-6-P + ADP}$
• EC 3 hydrolases: e.g., $\ce{AB + H2O -> A-OH + BH}$
  • EC 3.1 esterase: $\ce{ester + H2O -> acid + alcohol}$
    • nuclease: nucleid acid hydrolysis
      • cleave phosophodiester bonds
      • ribonuclease (RNase) -> cleave RNA
      • deoxyribonuclease -> cleave DNA
    • lipase: fat hydrolysis
    • EC 3.1.3 phosphatases: A-P + H2O -> A-OH + H-P
      • reverse of kinase?
      • EC 3.1.3.9 glucose-6-phosphatase
  • EC 3.2 glycosylases
    • cleave polysaccharide bonds
    • amylase: starch hydrolysis
    • chitinase
    • galactosidase
    • lactase
    • maltase
    • sucrase
  • EC 3.4 protease=peptidase
    • cleave polypeptide bonds (proteolysis)
    • $\ce{-NH-CH2-CO-NH-CH2-COO- + H2O -> -NH-CH2-COO- + NH3+-CH2-COO-}$
      • $\ce{H2O}$ splits into $\ce{H2+, O-}$ instead of $\ce{H+, OH-}$
• EC 4 lyases
  • break covalent bond
  • not using oxidation (else oxidoreductase)
  • not using water (else hydrolase)
  • EC 4.1.1.1 pyruvate decarboxylase
    • $\ce{pyruvate + H+ -> acetaldehyde + CO2}$
• EC 5 isomerases: $\ce{ABC -> BCA}$
  • move functional group within molecule
    • outside would be transferase
• EC 6 ligases: $\ce{A + B + ATP -> AB + ADP + P}$
  • use ATP to form covalent bond
  • cf. ligand
    • Latin: ligare = to connect
• [EC 7 translocases]

Enzyme kinetics

• also see notebook
• reaction speed $v$
  • depends on
    • temperature
      • constant in humans
    • $[E]$
    • E activity
    • $[S]$ <--
• Michaelis-Menten kinetics
  • Michaelis-Menten constant $K_m$
    • $[K_m] = \pu{mol / l}$
    • measure for E-S affinity
    • often near physiological [S]
      • so small changes have big effect
  • speed $v = \dfrac{V_{max} [S]}{K_m + [S]}$
    • $[v] = \pu{mol/s}$
    • $v$ vs $[S]$ plot
    • proof: see practicum 4
    • $[S] \ll K_m \iff v \approx \dfrac{V_{max}}{K_m} [S]$
      • initially linear
    • $[S] \gg K_m \iff v \approx V_{max}$
      • levels off towards horizontal asymptote
    • $[S] = K_m \iff v = \dfrac{1}{2} V_{max}$
  • $V_{max} = k_{cat} [E_{tot}] \iff k_{cat} = \dfrac{V_{max}}{[E_{tot}]}$
    • $[k_{cat}] = \pu{s-1}$
    • molecules (not moles!) converted per second for a single enzyme
    • less enzymes, lower $V_{max}$
• Lineweaver-Burk plot
  • linear
  • $\dfrac{1}{v}$ vs $\dfrac{1}{[S]}$
  • $\dfrac{1}{v} = \dfrac{K_m}{V_{max}} \dfrac{1}{[S]} + \dfrac{1}{V_{max}}$
• Eadie-Hofstee plot
  • linear
  • $\dfrac{v}{[S]}$ vs $v$
  • $\dfrac{v}{[S]} = \dfrac{V_{max}}{K_m} - \dfrac{1}{K_m}v$
• Hanes-Woolf
  • $\dfrac{[S]}{v} = \dfrac{K_m}{V_{max}} + \dfrac{1}{V_{max}}[S]$

Enzyme regulation

• enzyme inhibitors
  • irreversible due to covalent bonds
    • aspirine
    • penicillin
    • nerve gas (sarin, novichok): deactivate ACh-esterase
    • but not all covalent bonds are irreversible
      • e.g. phosphorylation
  • reversible: non-covalent bonds
    • competitive
      • inhibitor binds on active site where substrate usually binds
      • same $V_{max}$
      • higher/worse $K_m$
      • example: ethanol
        • inhibits alcohol dehydrogenase breaking down ethylene glycol/methanol
    • non-competitive
      • inhibitor binds on different site
      • same $K_m$
      • lower $V_{max}$
• allosteric control
  • Greek: allosteric = other shape
  • enzyme with 2 forms: high and low affinitiy
  • inhibition
    • often multi-subunit: catalytic and regulatory subunit
    • often first enzyme of cascade -> feedback inhibition
    • competitive or non-competitive
      • all non-competitive inhibitors are allosteric
  • activation
• cooperativity
  • positive or negative
  • e.g. myoglobin and hemoglobin
    • oxygen saturation vs oxygen partial pressure: sigmoid curve
    • opens up when more oxygen bound -> positive
• covalent modifications
  • phosphorylation
    • binding one or more P can activate some enzymes
    • e.g., glycogen phosphorylase a and b (in cytosol)
  • e.g., protease activation in small intestine via trypsin + enterokinase
• misc
  • RNA catalysis: ribozymes

H9 Glycolysis and fermentation

• adenosine triphosphate (ATP)
  • A-P~P~P
    • 1 phosphoester bond
      • $\Delta G^{0\prime} = \pu{-3.6 kcal//mol}$
    • 2 phosphoanhydride bonds
      • $\Delta G^{0\prime} = \pu{-7.3 kcal//mol}$
        • real life: [ATP] 5:1 [ADP]
        • $\pu{-14 kcal//mol} \leq \Delta G^{\prime} \leq \pu{-10 kcal//mol}$
  • hydrolysis: $\ce{ATP^4- + H2O -> ADP^3- + H+ + P_i^2-}$
    • $\ce{P_i = HPO4^2-}$
    • reverse: condensation
  • energy rich bond
    • = highly exergonic hydrolysis
    • charge repulsion between P groups
    • resonance stabilization
      • cf. $\ce{COO-}$
      • spread over 4 atoms in $\ce{P_i}$
        • optimal, low energy
      • when bound: spread over 3, higher energy
    • $\Delta S > 0$
      • spatial randomization
      • $\ce{ADP, P_i}$ more soluble
• oxidation = dehydrogenation (in organic chemistry)
  • oxidation: donate electron
  • typically by donating entire hydrogen atom
  • $\ce{2H+ + 2e- = 2H}$
  • always in pairs
  • $\ce{H-C-OH -> C=O + H+ + H-}$
    • extract proton ($\ce{H+}$) + hydride ion ($\ce{H-}$)
• reduction = hydrogenation
• nicotinamide adenine dinucleotide ($\ce{NAD+}$)
  • co-enzyme
  • electron acceptor
  • $\ce{NAD+ + 2H ->[reduction] NADH + H+}$
  • contains 1 adenosine as in RNA
    • AMP
  • other ribose bound to nicotinamide
    • nicotinamide can be reduced by hydrogenation
  • pyrophosphate bridge: $\ce{P_i}$s linked to each other
    • not to riboses as in DNA/RNA

Glycolysis

• in cytosol
• anaerobic
• partial oxidation of glucose to pyruvate
• abbreviations
  • G6P = glucose-6-P
  • F6P = fructose-6-P
  • F1,6BP = fructose-1,6-bisP
  • DHA = dihydroxyacetone (ketotriose)
  • DHAP = DHA phosphate
  • GA3P = glyceraldehyde-3-P
  • 1,3-BPG = 1,3 bisP-glycerate
  • 3-PG = 3-P-glycerate
  • 2-PG = 2-P-glycerate
  • PEP = Penol-pyruvate

Step	Enzyme	Abbr	Class
Gly-1	hexokinase		EC 2.7.1.1 (phospho)transferase
Gly-2	phospho-gluco-isomerase	GPI	EC 5.3.1.9 isomerase
Gly-3	phospho-fructokinase-1	PFK-1	EC 2.7.1.11 (phospho)transferase
	phospho-fructokinase-2	PFK-2	EC 2.7.1.x (phospho)transferase
Gly-4	aldolase		EC 4.1.2.13 lyase
Gly-5	triose phosphate isomerase	TPI	EC 5.3.1.1 isomerase
Gly-6	GA3P dehydrogenase	GAPDH	EC 1.2.1.12 oxidoreductase
Gly-7	phosphoglycerokinase	PGK	EC.2.7.2.3 (phospho)transferase
Gly-8	phosphoglyceromutase	PGM	EC 5.4.2.11 isomerase
Gly-9	enolase		EC 4.2.1.11 lyase
Gly-10	pyruvate kinase		EC 2.7.1.40 (phospho)transferase
E1	starch phosphorylase		EC 2.x.x.x transferase
E2	glycogen phosphorylase		EC 2.x.x.x transferase
E3	galactokinase		EC 2.7.1.6 (phospho)transferase
E4	uridyl transferase		EC 2.x.x.x transferase
E5	UDP-galactose epimerase		EC 5.1.3.2 isomerase
E6	UDP-glucose pyrophosphorylase		EC 2.x.x.x transferase
E7	phosphoglucomutase		EC 5.4.2.2 isomerase
E8	lactase		EC 3.2.1.108 hydrolase
E9	maltase		EC 3.2.1.20 hydrolase
E10	sucrase		EC 3.2.1.x hydrolase
E11	hexokinase		EC 2.7.1.1 (phospho)transferase
E12	phosphomannoisomerase		EC 5.x.x.x isomerase
E13	glycerokinase		EC 2.7.1.30 (phospho)transferase
E14	glycerol-3-phosphate dehydrogenase		EC 1.1.1.8 oxidoreductase
Gng-1	glucose-6-phosphatase	GPase	EC 3.1.3.9 hydrolase
Gng-3	fructose-1,6-bisphosphatase	FBPase	EC 3.1.3.11 hydrolase
Gng-10	pyruvate carboxylase	PC	EC 6.4.1.1 ligase
Gng-10	phosphoenolpyruvate carboxykinase	PEPCK	EC 4.1.1.32 lyase
FERM	lactate dehydrogenase	LDH	EC 1.1.1.27 oxidoreductase
FERM	pyruvate decarboxylase	PDC	EC 4.1.1.1 lyase
FERM	alcohol dehydrogenase	ADH	EC 1.1.1.1 oxidoreductase

• 10 step process
  • $\ce{glucose + 2ATP + 2ADP + 2P_i + 2NAD+ -> 2 pyruvate + 4 ATP + 2NADH + 2H+}$
    • problem if $\ce{NAD+}$ supply is depleted
    • recover in later stage (fermentation or aerobic respiration/ETS)
  • phase 1 (Gly1 - Gly5)
    • cleave sugar by double phosphorylation
    • $\ce{glucose + 2ATP -> 2 {GA3P} + 2ADP}$
    • Gly-1: $\ce{G + ATP ->[hexokinase] {G6P} + ADP}$
      • hexokinase = kinase for hexoses
      • ATP: energy + P donor
    • Gly-2: $\ce{{G6P} <=>[GPI] {F6P}}$
      • aldose-ketose isomerase
      • using acid-base catalysis
        • move $\ce{H}$s around
    • Gly-3: $\ce{{F6P} + ATP <=>[PFK-1] {F1,6BP} + ADP}$
    • Gly-4: $\ce{{F1,6BP} <=>[aldolase] DHAP + {GA3P}}$
      • split hexose in two trioses
        • DHA: ketotriose
        • GA: aldotriose
    • Gly-5: $\ce{DHAP <=>[TPI] {GA3P}}$
      • aldose-ketose isomerase
      • using acid-base catalysis
        • cf. Gly-2
  • phase 2 (Gly6 - Gly7)
    • $\ce{2 {G3P} + 2P_i + 2ADP + 2NAD+ -> 2 3-PG + 2ATP + 2NADH}$
    • count everything double from here on
    • Gly-6: $\ce{2{GA3P} + 2P_i + 2NAD+ <=>[GAPDH] 2 1,3-BPG + 2NADH + 2H+}$
      • dehydrogenation of aldehyde
      • using Cys with $\ce{E-SH}$ group
        • "covalent catalytic intermediary"
      • create energy rich bond ~
        • $\ce{R-(C=O)\bond{~}O-P}$
    • Gly-7: $\ce{2 1,3-BPG + 2 ADP <=>[PGK] 2 3-PG + 2 ATP}$
  • phase 3 (Gly8 - Gly10)
    • $\ce{2 3-PG + 2ADP -> 2 pyruvate + 2ATP}$
    • Gly-8: $\ce{2 3-PG <=>[PGM] 2 2-PG}$
    • Gly-9: $\ce{2 2-PG <=>[enolase] 2 PEP + 2 H2O}$
    • Gly-10: $\ce{2 PEP + 2 ADP + 2H+ ->[pyruvate kinase] 2 pyruvate + 2 ATP}$
      • Gly-10a: $\ce{2 PEP + 2 ADP + 2H+ <=>[pyruvate kinase] 2 enolpyruvate}$
      • Gly-10b: $\ce{2 enolpyruvate -> 2 pyruvate}$
      • enolpyruvate is unstable
  • all steps except 1, 3, 10 are reversible
  • steps 1,3,7,10 involve phosphokinases
    • steps 1,3 require ATP
    • steps 7,10 yield ATP
  • steps 2,5 uses acid-base catalysed aldose-ketose isomerase
  • step 4 splits the hexose -> lyase
  • steps 5,8 move P around -> isomerases
  • step 6 is actual oxidation -> oxidoreductase, using Cys E-SH
  • types of enzymes
    • kinases = transferases: 4
    • isomerases: 3
    • lyases: 2 (aldolase, enolase)
    • oxido-reductase: 1 (GAPDH)
• alternative substrates
  • monosaccharides
    • glucose -> G6P -> F6P
    • fructose -> F6P
    • mannose -> M6P -> F6P
    • galactose -> Ga1P -> ... -> G1P -> G6P -> F6P
  • hydrolize disaccarides
    • humans always hydrolise
    • bacteria can cleave by phosphorylation
      • cf. polysaccharides below
      • sucrose -> G6P + F
      • more efficient (saves 1 ATP)
  • phosphorylate polysaccharides to split of G1P
    • only path where no initial ATP is required
    • glycoside bonds release enough energy when hydrolysed
  • always ATP required to phosphorylate each monosaccharide
    • exception: polysaccharides
  • glycerol
    • $\ce{glycerol + ATP ->[glycerokinase] glycerol-3-P + ADP}$
    • $\ce{glycerol-3-P + NAD+ ->[{Gly3PDH}] DHAP + NADH + H+}$

• gluconeogenesis
  • in liver
  • start from lactate or pyruvate
  • reverse of glycolysis
  • except for 3 irreversible steps (1,3,10)
    • Gng-1: $\ce{{G6P} + H2O ->[GPase] glucose + P_i}$
    • Gng-3: $\ce{{F1,6BP} + H2O ->[FBPase] {F6P} + P_i}$
    • Gng-10
      • $\ce{2 pyruvate + 2CO2 + 2ATP ->[PC] oxaloacetate + 2ADP}$
      • $\ce{oxaloacetate + 2GTP ->[PEPCK] PEP + 2GDP + 2CO2}$
      • expensive step

• Cori cycle
  • glycolysis and fermentation in muscles
    • $\ce{glucose + 2NAD+ -> 2 pyruvate + 2ATP + 2NADH}$
    • $\ce{2 pyruvate + 2 NADH -> 2 lactate + 2 NAD+}$
  • gluconeogenesis in liver
    • $\ce{2 lactate + 2NAD+ -> 2 pyruvate + 2NADH}$
    • $\ce{2 pyruvate + 6ATP + 2NADH -> glucose + 2NAD+}$
  • $\ce{NAD+}$ neutral operations on both sides
  • gain 2 ATP, lose 6 ATP

Fermentation

• anaerobic
• recovers $\ce{NAD+}$
• works for most sugars except pentoses and lactose
• two options
  • pyruvate -> lactate
    • pH decreases -> enzyme activity decreases
    • $\ce{pyruvate + NADH + H+ ->[LDH] lactate + NAD+}$
    • LDH = lactate dehydrogenase = oxidoreductase
  • pyruvate -> ethanol + CO2
    • $\ce{pyruvate + H+ ->[PDC] acetaldehyde + CO2}$
    • $\ce{acetaldehyde + NADH + H+ ->[ADH] ethanol + NAD+}$
    • PDC = pyruvate decarboxylase = EC 4.1.1.1 lyase
    • ADH = alcohol dehydrogenase = oxidoreductase
    • 17g sugar -> 1% alcohol
• efficiency of glycolysis thus far (under standard conditions)
  • $\ce{C6H12O6 + 6O2 -> 6CO2 + 6H2O}$
    • $\Delta G^{0\prime} = \pu{-686 kcal//mol}$
  • $\ce{lactate + 3O2 -> 3CO2 + 3H2O}$
    • $\Delta G^{0\prime} = \pu{-319.5 kcal//mol}$
  • $\dfrac{2 \cdot 319.5}{686} \approx 93\%$ of free energy in glucose remains after glycolysis
  • free energy consumed
    • $\pu{686 kcal//mol} - 2 \cdot \pu{319.5 kcal//mol} = \pu{47 kcal//mol}$
  • energy created: 2 ATP
    • $2 \cdot \pu{7.3 kcal//mol} = \pu{14.6 kcal//mol}$
  • efficiency: $\dfrac{14.6}{47} \approx 31\%$
    • 2% of total free energy in glucose

Regulation

• allosteric regulation
• affects irreversible steps 1,3,10
• Gly-1: hexokinase
  • G6P -
• Gly-3: PFK-1
  • F2,6BP +
  • AMP +
  • ATP -
  • citrate -
• Gng-3: FBPase
  • F2,6BP -
  • AMP -
• Gly-10: pyruvate kinase
  • F1,6BP +
  • ATP -
  • Acetyl CoA -
• Gng-10: PC
  • Acetul Coa +

H10 Aerobic respiration

• in mitochondria
  • cf. glycolyse in cytosol
  • exception: in membrane+cytosol for bacteria

Mitochondria

• mitochondria
  • outer membrane (7nm)
    • contains porines
      • max 5 kDa: glucose, ATP, protein <50 AA
      • made from $\beta$ sheets
  • intermembrane space
    • lots of H+
    • pH - 1
  • inner membrane
    • cristae
      • increase inner membrane surface
  • matrix
    • (circular) DNA + ribosomes
  • outer+inner membrane connect sometimes
• goals
  • complete breakdown of pyruvate
    • 38 ATP
  • recover NAD+
• pyruvate flow
  • through porines of outer membrane
  • transport through inner membrane
• phase 1: pyruvate to Acetyl CoA
  • in matrix
  • oxidative decarboxylation: remove CO2
  • CoA = co-enzyme A
    • ~= AMP + pantothenic acid (vitamin B5)
    • HS-CoA vs Acetyl CoA
      • sulfhydryl vs thio-ester
  • $\ce{pyruvate + HS-CoA + NAD+ ->[PDH] AcetylCoA + NADH + H+ + CO2}$
    • once for each pyruvate
    • thiamine = vitamin B1
    • two irreversible steps
      • add $\ce{HC#thiamine}$
      • release $\ce{CO2}$
        • 3C -> 2C
    • two reversible steps
      • oxidation with $\ce{NAD+ <-> NADH + H+}$
        • create energy rich bond ~
      • $\ce{HC#thiamine <-> HS-CoA}$
    • pyruvate dehydrogenase (PDH)
      • inactivate when phosphorylated
      • $\ce{PDH-P + H2O ->[PDH phosphatase] PDH + P_i}$
      • $\ce{PDH + ATP ->[PDH kinase] PDH-P + ADP}$
      • regulation
        • down when plenty of energy or products
          • ACoA
          • NADH
          • ATP
        • up when low on energy or many substrates
          • CoA
          • NAD+
          • AMP

Citric acid cycle

• phase 2: citric acid cycle = Krebs = TCA
  • in matrix
  • once for each pyruvate/ACoA
  • $\ce{ACoA + 3NAD+ + FAD + ADP + P_i -> 2CO2 + 3NADH + FADH2 + HS-CoA + ATP}$
  • common molecules
    • oxaloacetate: 4C
    • acetyl CoA: 2C
    • citrate: 6C
    • alpha-ketoglutarate: 5C
    • succinyl CoA: 4C
    • succinate: 4C
    • fumarate: 4C
    • malate: 4C
    • 4 + 2 = 6
    • 6 - 1 = 5
    • 5 - 1 = 4
  • 8 steps (each individually irreversible)
  • CAC-1: $\ce{oxaloacetate + ACoA + H2O ->[CS] citrate + HS-CoA + H+}$
    • combinating decreases $S$
    • must be compensated with $E$ from ~
  • CAC-2: $\ce{citrate ->[aconitase] isocitrate}$
    • switch H and OH group
  • CAC-3: $\ce{isocitrate + NAD+ ->[ICDH] \alpha-ketaglutarate + CO2 + NADH + H+}$
  • CAC-4: $\ce{\alpha-ketaglutarate + HS-CoA + NAD+ ->[KGDH] succinyl CoA + CO2 + NADH + H+}$
    • cf. pyruvate -> ACoA
  • CAC-5: $\ce{succinyl CoA + GDP + P_i ->[SCS] succinate + HS-CoA + GTP}$
    • GTP used to ADP->ATP
    • succinate = barnsteenzuur
  • CAC-6: $\ce{succinate + FAD ->[SDH] fumarate + FADH2}$
    • remove 2H, add double bond
    • fumarate: $\ce{COO- -CH=CH-COO-}$: highly dehydrogenated
    • FAD = flavin adenine dinucleotide
      • co-enzyme
      • cf. NAD+
      • AMP + P + riboflavin
      • riboflavin
        • ~vitamin B
        • 3 6-rings
        • oxidized (without H) or reduced ($\ce{FADH2}$)
  • CAC-7: $\ce{fumarate + H2O ->[FH] malate}$
    • malate = appelzuur
  • CAC-8: $\ce{malate + NAD+ ->[MDH] oxaloacetate + NADH + H+}$
  • regulation
    • always during oxidation=dehydrogenation with NAD+ (not FAD)
    • CAC-3: ICDH
      • NADH -
      • ADP +
    • CAC-4: KGDH
      • NADH -
      • Succinyl CoA -
    • CAC-8: MDH
      • NADH -
  • summary
    • oxidative decarboxylation in CAC-3,4
    • single ATP gained in CAC-5
    • CAC-6 uses FAD instead of NAD+
    • 2 CO2 are actually released from $\ce{COO-}$ of citrate, not from ACoA
    • enzym classes
      • 4 oxidoreductases = dehydrogenases (5 when including PDH)
      • 2 lyase
      • 1 transferase?
      • 1 ligase
    • $\ce{glucose + 10NAD+ + 2FAD + 4ADP + 4P_i -> 6CO2 -> 10NADH + 2FADH2 + 4ATP}$

Step	Enzyme	Abbr	Class
	pyruvate dehydrogenase	PDH	EC 1.2.4.1 oxidoreductase
CAC-1	citrate synthase	CS	EC 2.3.3.1 transferase (former lyase?)
CAC-2	aconitase		EC 4.2.1.3 lyase
CAC-3	isocitrate dehydrogenase	ICDH	EC 1.1.1.42 oxidoreductase
CAC-4	ketoglutarate dehydrogenase	KGDH	EC 1.x.x.x oxidoreductase
CAC-5	succinyl CoA synthetase	SCS	EC 6.2.1.4 ligase
CAC-6	succinate dehydrogenase	SDH	EC 1.3.5.1 oxidoreductase
CAC-7	fumarate hydratase	FH	EC 4.2.1.2 lyase
CAC-8	malate dehydrogenase	MDH	EC 1.1.1.37 oxidoreductase

• note: enzyme mostly contains name of substrate, rarely product
  • exceptions: CAC1,2

Fat metabolism

• triglyceride = glycerol + fatty acid
  • $\ce{glycerol + ATP + NAD+ -> DHAP + ADP + NADH + H+}$ in glycolysis
    • 2 steps
  • 2nC fatty acids -> $\beta$-oxidation to n ACoA -> CAC -> n ATP
    • in matrix
    • $\alpha$ = head of fatty acid = $\ce{-CH2-COO-}$
    • $\beta$ = $\ce{-CH2-CH2 -}$ right after head
      • gets oxidized to $\ce{-CH2-(C=O) -}$ to form new head later
    • FA-1: activation
      • $\ce{fatty acid + ATP + HS-CoA -> fatty acyl CoA + AMP + PP_i}$
      • costs 2 phosphoanhydride bonds
      • creates high energy bond ~
        • does not get used immediately?
    • rest in n-1 cycles of 4 steps
      • remove 2C as ACoA every cycle
      • $\ce{fatty acyl CoA + FAD + H2O + NAD+ + HS-CoA -> fatty acyl CoA + ACoA}$
      • FA-2: oxidation (FAD)
      • FA-3: hydration (H2O)
      • FA-4: oxidation (NAD+)
      • FA-5: thiolysis
    • process can deplete free CoA when lots of fat is burned
      • halts CAC
      • fat -> ketone bodies (partial oxidation)
        • fat -> 2 ACoA -> Acetoacetyl CoA -> Acetoacetate
        • heart and brain can consume ketone bodies
      • can lower blood pH -> ketoacidosis -> health risk?

Protein metabolism

• protein -> AA
  • endoprotease and exoprotease
  • reuse for protein synthesis
  • or break down to generate ATP
    • AA -> alpha-keto acid + ureum
      • remove amino group
      • Ala <-> pyruvate -> CAC
      • Asp <-> oxaloacetate -> CAC
      • Glu <-> alpha-ketogluterate -> CAC
    • CAC is amphibolic: connects anabolic + catabolic

Misc

• glyoxylate cycle
  • fat -> sugar
  • only in plants
  • in glyoxosome
  • (triglycerides -> )ACoA -> succinate -> PEP -> sugar
  • details not important

Electron transport chain/system (ETS)

• inner membrane proteins
• reminder: oxidation = donating electrons
• $E'_0$: standard reduction potential (in Volt)
  • cf. standard conditions for $\Delta G'$
  • spontaneity of redox reaction
  • $E'_0 > 0 \implies$ e acceptor -> reduce
  • $E'_0 < 0 \implies$ e donor -> oxidize
  • $\Delta E'_0 = E'_{0, acc} - E'_{0, donor}$
  • $\Delta G^{0\prime} = -nF\Delta E'_0$
    • minus -> opposite signs
    • number of electrons $n$
    • Faraday constant $F \approx \pu{23 cal//mol V}$
• $\ce{NADH + H+ + 0.5O2 -> NAD+ + H2O}$
  • $E'_0(\ce{O2}) = +0.816V$
    • can be reduced: $\ce{0.5O=O + 2H+ + 2e- -> H-O-H}$
  • $E'_0(\ce{NADH}) = -0.32V$
    • reduced form, can be re-oxidized to $\ce{NAD+}$
  • $\Delta E'_0 = 0.816V - (-0.32V) = 1.136V$
  • $\Delta G^{0\prime} = -2 \cdot \pu{23 kcal//mol V} \cdot \pu{1.136V} = \pu{-52.4 kcal/mol}$
• $\ce{FADH2 + 0.5O2 -> FAD + H2O}$
  • $\Delta G^{0\prime} = \pu{-45.9 kcal/mol}$
• electron carriers
  • flavo (FAD, FMN): p + e
    • FMN = flavo mono nucleotide
  • Fe-S: only e
  • cytochrome (heme): only e
  • Cu cytochrome: only e
  • coenzym Q (CoQ) = ubiquinon: p + e
    • not a protein
    • 6-ring with =O at top and bottom
    • CoQ <-> CoQH <-> CoQH2
    • acts as pump
      • $\ce{O + H+ + e- <-> OH}$ on inside
      • $\ce{OH <-> O + H+ + e-}$ on outside
  • four respiratory complexes
    • 
    • goal: increase $E'_0$ towards $E'_0(\ce{O2})$ step by step
    • organized in supercomplexes = respirasomes
    • Q cycle not important
    • complex I: NADH-CoQ ORase
      • $\ce{NADH + 2e- -> FMN + NAD+ + H+ -> Fe-S -> CoQH2}$
      • complex I pumps $\ce{4H+}$
    • complex II: succinate-CoQ-ORase
      • alternative to complex I with FAD instead of NADH
      • does not pump
    • complex III: CoQ-cytochrome c ORase
      • $\ce{CoQH2 -> Fe-S -> Cyt c_1 -> Cyt c}$
      • CoQ pumps $\ce{2H+}$
      • complex III pumps $\ce{2H+}$
      • total: $\ce{4H+}$ from matrix to intermembrane space
    • complex IV: cytochrome c oxidase
      • $\ce{Cyt c -> Cyt a -> Cyt a_3 -> Fe-Cu}$
      • $\ce{0.5O2 + 2H+ + 2e- -> H2O}$
      • complex IV pumps $\ce{2H+}$
      • cyanide and azide could bind $\ce{Fe-Cu}$ and block this process
    • incomplete reduction of O2
      • if complex I or III transfer e to O2
        • -> toxic superoxide anion $\ce{O2-}$
        • or $\ce{H2O2}$
    • result
      • NADH oxidation: 4+4+2=10H+
      • FAD oxidation: 4+0+2=6H+
• $F_0 F_1$ ATP synthase
  • 
  • also in inner membrane
  • proton channel: intermembrane space -> matrix
  • converts 1 ADP -> 1 ATP per 3-4 protons
  • proton motive force (pmf)
    • $pmf = V_m + 2.303 RT \frac{\Delta pH}{F}$
    • $V_m = 0.16V$
    • $\Delta pH = 1$
    • $T = 310K$
    • $pmf = 0.22 V$
    • $\Delta G^{0\prime} = -nF(pmf)$
      • dus $pmf = E'_0$?
  • $F_0$: proton translocator
    • subunit a x1
      • channel
    • subunit b x2
      • stalk connecting F0 with F1
    • subunit c x10
      • 1 of 10 has ionic bond with a (Asp-Arg)
      • proton neutralizes Asp
      • rotates 1/10 per proton
      • turns $\gamma$
  • $F_1$: create ATP from proton gradient
    • subunit $\alpha$ x3
      • forms $\alpha_3\beta_3$ hexagon
    • subunit $\beta$ x3
      • ATP synthesis
      • 3 conformations
        • open
        • loose
        • tight: ADP + P -> ATP
          • $\Delta G^{0\prime} \approx 0$ instead of $\pu{+7.3 kcal//mol}$
    • subunit $\delta$ x1
      • link b with $\alpha_3\beta_3$ hexagon
    • subunit $\gamma$ x1
      • stalk
      • rotates to transmit energy from F0 to F1
    • subunit $\varepsilon$ x1
      • links $\gamma$ to c-ring
      • also rotates
• uncouplers
  • e.g., dinitrophenol (DNP) or thermogenine
    • bind with protons -> no gradient
    • transfer back across membrane
  • oxygen still consumed
  • but no ATP synthesis
• final result per glucose
  • $\ce{10NADH + 10H+ + 2FADH2 + 6O2 + 34ADP + 34P_i -> 10NAD+ + 2FAD + 12H2O + 34ATP}$
  • 34 is best case scenario
    • alternative: NADH -> shuttle
      • -> liver, heart, kidney: 3 ATP
      • -> muscle, brain: 2 ATP
    • pmf can also be used elsewhere
  • $\ce{C6H12O6 + 6O2 + 38ADP + 38P_i -> 6CO2 + 6H2O + 38ATP}$
  • total: 36-38ATP
  • ATP hydrolysis: 10 - 14 kcal/mol
    • let's say 10
    • times 38 -> 380 kcal/mol
    • compare with glucose: $\Delta G^{0\prime} = \pu{-686 kcal/mol}$
    • ~55% efficiency
• result for other inputs
  • NADH ->[ETS] 3 ATP
  • FADH2 ->[ETS] 2ATP
  • ACoA ->[TCA] 1 ATP + 3 NADH + 1 FADH2 ->[ETS] 12 ATP
• notes
  • GTP in 3 places
    • Gng-10: oxaloacetate -> PEP
    • CAC-5: succinyl-CoA -> succinate
    • (later) importin/exportin with RAN
  • FAD in 2 places
    • beta oxidation: first of two oxidations
    • CAC: succinate -> fumerate
  • ligase enzyme in 2 places
    • Gng-10: puryvate carboxylase to create oxaloacetate
    • CAC: succinyl CoA synthetase
• TODO create Excel of ATP per substance like in WZ3

H11 Photosynthesis

• endosymbiosis (like mitochondria)
• in chloroplast
  • stroma
  • thylakoid
    • membrane ~= inner membrane mitochondria
    • lumen ~= intramembrane space mitochondria -> H+ gradient
    • stack = granum
• two processes
  • transduction: light -> chemical energy
    • photosystem I and II
      • coupled by electron transport chain
    • electron source: water
    • electron acceptor: $\ce{NADP+}$, not $\ce{NAD+}$
    • F0F1 ATPase
  • create carbohydrates from from CO2 and H2O
    • Calvin cycle
      • ribulose

H16 DNA and chromosomes

16.1 Genetic material

• experiment: pneumococcus R <-> S in mice
  • heat killed S + living R -> living S -> dead
  • protease -> no
  • RNAse -> no
  • lipase -> no
  • sacharase -> no
  • DNAse -> yes
• (bacterio)faag
  • lytische cyclus: vermenigvuldigen
  • lysogene cyclus: integreeg DNA in gastheer, inactief
  • experiment: $\ce{^35 S}$ vs $\ce{^32 P}$
    • -> DNA
    • niet in mantel
    • wel in sommige kopieen
    • S in proteinen (Cys of Met), dus enkel in mantel
  • RNA faag
    • Tobacco Mosaic Virus (TMV)
    • Ribgrass Mosaic Virus (RMV)
    • RNA TMV + mantel RMV = TMV
  • retrovirus
    • HIV
      • 2x (+)ssRNA?
    • reverse transciptase: RNA -> DNA
  • +ssRNA
    • TMW
    • COVID-19
  • -ssRNA
    • influenza
  • dsDNA
    • HPV

16.2 DNA structure

• TODO
• see handwritten notes

16.3 DNA packing

• TODO
• see handwritten notes

16.4 Nucleus

• TODO
• see handwritten notes

H17 DNA replication and error correction

• TODO
• see handwritten notes

H18 transcriptie

• TODO
• volgorde mRNA maturatie
  • transcriptie initiatie (reminder: 5'->3')
  • toevoegen van 5’ cap
  • pre-mRNA-splicing
    • introns weg
  • toevoegen van poly-A staart
  • transport naar het cytoplasma

H19 translatie

• TODO

• voorbeelden van hoe cel omgaat met nonsense/nonstop mutatie in mRNAs
  • (1) suppressor tRNAs
    • bv. nonsense suppressor tRNA
  • (2) nonsense gemedieerde mRNA afbraak (NMD)
  • (3) nonstop mRNA decay

• energieverbruik (fosfoanhydride) translatie
  • initiatie
    • prokaryoot: 3
    • eukaryoot: 4
  • elongatie: 4/AZ
    • ATP -> AMP
    • 2GTP -> 2GDP
    • extra bij fouten
  • terminatie: 1

H20 regulatie

• TODO

H21 methods

• TODO
• blots
  • southern: DNA
  • northern: RNA
  • western: protein

H24 mitose

• mitose (2n/4C -> 2n/2C)
  • profase
    • chromosomen
    • 2xMTOC met elk 2 centriolen
  • prometafase
    • microtubuli
      • kinetochoor -> chromosomen
      • polair (pool-pool)
      • astraal (pool-membraan)
  • metafase
    • chromosomes in metaphase plate
  • anafase
    • afbraak cohesin
    • anafase A)
      • kinetochoor MTs korter
        • kinesine aan + (chromosoom) uiteinde
        • ander kinesine aan - (centrosoom)
      • zusterchromatiden uit elkaar
    • anabase B)
      • polaire MTs langer
        • polen uit elkaar
        • bipolaire kinesine
    • astrale MTs korter
      • dyneinen
  • telofase
    • chromosoom decondensatie
    • nucleoli verschijnen
    • kernmembraan wordt gevormd
    • begin cytokinese
  • cytokinese
    • contractiele actine-microfilamenten
    • contractiele ring: Interactie actine – myosine
      • myosine niet nodig voor mitose zelf
    • prokaryoten
      • FtsZ eiwit
• regulering mitose
  • cycline-afhankelijke eiwitkinasen (Cdk)
  • growth factors (GFs)
  • 3 checkpoints = transitiepunten
    • G1/S
    • S-fase: replicatie licensing
      • MCM (DNA helicase) complex verwijderd
      • S-fase Cdk -> fosforylering van ORC + helicase laders -> max 1 copy
      • geminine
    • G2/M
    • meta/ana
      • anafase-promoting complex (APC)
• apoptosis
  • != necrose
  • caspase
  • Bcl2 (anti-apoptosis) inhibited
  • DNA damaage -> p53 -> inhibit Blc2
  • GF -> ... BAD -> inhibit Bcl2

H25 meiose

• asexual reproduction
  • parthenogenese: 1 unfertilized gamete -> embryo
• meiose
  • 1 diploid -> 4 haploid
  • meiose I (2n/4C -> 1n/2C)
    • pro I
      • leptotene
      • zygotene
        • start homologous chromosomes synapsis
          • "bivalent"
          • between non-sister chromatids
          • non-uniform -> hotspots
      • pachytene
        • start crossing over
      • diplotene
        • chiasmata due to crossing over shows
          • achiasmy: in male fruit flies
        • synaptonemal complex dissolves
      • diakinesis
    • meta I
    • ana I
      • scheiding homologen
        • bv. X-Y uit elkaar
        • zusterchromatiden nog wel samen
          • shugoshin -> geen afbraak cohesin
    • telo I
    • cytokinese
      • two haploid daughter cells
  • interfase zonder S-fase
  • meiose II (1n/2C -> 1n/1C)
    • pro II
    • meta II
    • ana II
    • telo II + cytokinesis
  • non-disjunction
    • euploid: OK
    • aneuploid
      • -somie
        • 1 chromosoom
        • monosomie
        • trisomie
          • 21 -> Down
        • XXY: Klinefelter
        • XYY
        • XXX
        • X: Turner
      • polyploid
        • alle chromosomen
        • triploid (3n)
        • tetraploid (4n)
• oogenese
  • 4 haploid
    • 1 eicel
    • 3 poollichamen
    • pauze pro I (foetus 7md)
    • meta II arrest (tot bevruchting)
• Mendel
  • gen met 2 varianten (= allelen)
  • homozygoot (YY of yy)
  • heterozygoot (Yy)
  • dominant (Y)
  • recessief (y)
  • genotype vs fenotype
  • Punnett square
    • sikkelcel: recessief, 1/4 kans
• recombinatie
  • cf. H17
  • Spo1: endonuclease voor dsDNA
  • Rad51: ssDNA ligase
  • bronnen variatie
    • pro I: crossing over
    • ana I: splitsing homologen
  • types in bacteria
    • translation
    • transduction by phage
    • conjugation by sex pilus + F plasmid

H26 kanker

• TODO

Practicum 1 - fotometrische dosage van eiwitten

• wet Lambert-Beer
  • absorbantie $A$
  • laagdikte $B$
  • concentratie $C$ [mg/ml]
  • intredend licht $I_0$
  • uittredend licht $I_t$
  • extinctiecoefficient $k$
    • karakteristiek voor product
    • mate van absorptie voor bepaalde golflengte
    • hier: $\lambda = \pu{540 nm}$
    • $f(\lambda)$
  • $A = \log \frac{I_o}{I_t} = kBC = \alpha C$
    • $I_o = I_t \implies A=0$
    • $I_t$ stijgt -> $A$ daalt
    • linear verband zwakt af bij hogere concentraties

Practicum 2 - DNA: structuur, amplificatie en restrictiedigestie, gelelektroforese

• structuur: zie theorie
• amplificatie
  • PCR
  • materiaal
    • template DNA om te vermenigvuldigen
    • primers: ssDNA
    • dNTPs: bouwstenen (dATP, dTTP, dCTP, dGTP)
    • Taq DNA polymerase (hitteresistent)
    • buffer
  • cyclus (elke 90s)
    • DNA denaturatie
      • verwarm tot 94°C
    • primer hybridisatie / annealing
      • verlaag temp tot 58°C
      • primers binden met template DNA
    • DNA elongatie
      • verhoog tot 72°C
      • polymerase bouwt het DNA vanaf 3' van primers verder op
  • herhaal $N$ keer
    • practicum: 25
    • praktijk: veel meer
    • aantal kopieen: $2^N$
• restrictie-digestie
  • doel: knip DNA op specifieke plaats
  • a.h.v. restrictieenzymen = endonucleasen
  • op 37°C
  • hier: knip dsDNA in (circulair) plasmide (pGem-T)
• gelelektroforese
  • doel: lengte DNA fragmenten bepalen
  • agarose gel
    • niet geladen
    • polair
    • poeder + buffer -> koken -> afkoelen -> polymeriseren, crosslinks
  • elektro -> elektrisch veld
    • DNA negatief -> gaat naar positieve pool
    • korter -> minder hinder door aragose -> verder
    • laadbuffer: hoge dichtheid + kleurstof om visueel verloop te volgen
    • DNA ladder: DNA met bekende lengte als controle
  • na 30min
    • SYBR Safe toevoegen
    • bindt aan DNA -> fluorescent bij UV licht

Practicum 3 - koolhydraten en lipiden

• reducerend vermogen van suikers (Benedict test)
  • oxidatie van aldohexose: $\ce{H-C=O -> HO-C=O}$
  • reductie
    • $\ce{Cu^2+ -> Cu+}$ (+II to +I)
    • $\ce{C6H12O6 + 2Cu^2+ + 2H2O -> C6H12O7 + Cu2O v + 4H+}$
  • test per suiker
    • glucose: ja
    • fructose: ja
    • sucrose: nee
    • lactose: ja
    • glycogeen: nee
• hydrolyse van disacchariden
  • zure hydrolyse: $\ce{HCl}$
    • herhaal test
      • sucrose -> glucose + fructose: ja
      • glycogeen: ?
  • enzymatische hydrolyse: bv. amylase voor zetmeel
    • skipped
• fermentatie
• extractie lipiden uit melk
  • vooral triacylglycerol
    • 2/3 verzadigd
  • volle melk: 3.5% vet
  • halfvolle melk: 1.5% vet

Practicum 4 - enzymkinetica

• theorie: H6 Enzymes
• zie notebook
• enzym: alkaline phosphatase
  • class EC 3.1.3.1 -> hydrolase

Practicum 5 - structuur macromoleculen

• toepassing theorie

Werkzitting 1

Werkzitting 2

Werkzitting 3

Werkzitting 4

Werkzitting 5

Werkzitting 6