Biochemistry Flashcards
Covalent bonds
Shared pairs of electrons - strong (200-800kJ/mol), defined length + direction
O-4, N-3, S-2, P-5.
Single bonds allow rotation, different conformations more energetically favourable than others.
Functional groups
Mols w/ carboxyl -CO groups are acids.
Amino groups +ve charged, phosphate groups -ve charge in neutral conditions
Non covalent bonds
Weaker (<30kJ/mol), unequal sharing of electrons between nuclei.
Inc. hydrogen, electrostatic, van der waals, hydrophobic
Differing electronegativity of elements causes polarity: F > O»_space;S = C > H.
S-H has moderate polar bond, C-H non-polar covalent
Hydrogen bonds
Most common dipole-dipole bond, acceptor must be around 1.8nm away + in straight line. 30kJ to break H bonds.
Water mol can form 4 H bonds w/ other mols, 2 as acceptor + 2 as donor.
Donors: amino group, hydroxyl group.
Acceptors: imine, ether, ketone
Electrostatic interactions
Between opposite charges, stringer than H bond + greater distances than other non-covalent bonds.
e.g. acidic + basic a. acids (glutamate + lysine) -> salt bridge or ion pair
Attraction can be weakened by screening of water mol/other ions on protein surface, pairs in hydrophobic interior stronger/more stable
Van der waals
Between permanent or inducible dipoles (C-H), depends strongly on distance.
Inducible dipoles come due to random asymmetry/fluctuations, sum of vdw radius is optimum distance for inducible dipole interaction, force v. small (< 1kJ/mol) - large numbers can stabilise mol e.g. DNA
Hydrophobic interactions
Not bonds, cant engage in H binding as few/no polar bonds in hydrophobic mols.
Increase in entropy, every CH2 group out of water increases entropy (s) by +3kJ/mol so its favourable.
Molecules in water
Water determines shape molecules assume. Is molecular dipole so forms H bonds w/ itself. Can disrupt NaCl lattice by forming hydration shell around ions.
Alanine (carboxyl + amino) very polarised so more soluble, can form H bonds w/ water (solvation), hydrophilic.
Benzene is non-polar + hydrophobic -> reduces entropy as water has reduced motility around it.
Phospholipids amphipathic/amphiphilic, can migrate into micelles/vesicles.
Titration curves
Can study pH of a solution of weak acid by gradually adding small vols of strong base - until molar equivalent reached.
Flattest parts of curve where pH = pKa -> strongest buffering capacity e.g. buffers normally have pK of 6.5-7.5
A. acids (esp glycine) have acidic carboxyl & basic amino so titration curve has 2 pKa values -> amphoteric/amphiprotic.
If pKa > pH then more acid then base, vise versa
Amino acid structure
Zwitterion - under normal conditions have + and - charge.
C is chiral asymmetric. Enantiomers are stereoisomers that are mirror images of each other.
If carboxyl on left then it is L, if on right then it is D enantiomer.
-> mainly L amino acids in proteins
Glycine only a. acid without enantiomeric form (not chiral)
Fischer projection
Positions carboxyl group at top, R group at bottom of central C atom.
If amino group on left it is an L enantiomer, if on right then it is a D enantiomer.
Aliphatic a. acids
Non-aromatic hydrocarbon side chains.
G, A, V, L, I -> V to I have branched side chain
More hydrophobic as you go along.
Proline
Aliphatic a. acid (3C chain) BUT has heterocycle, much less hydrophobic + rotation restricted around N-C
Aromatic a.acids
Ring structures w/ alternating double bonds (delocalised pi electrons), varying hydrophobicity.
F, Y, W, H
-> H can have +ve charge, is a weak acid pKa =6
Both H & W engage in H bonding
Hydroxyl a. acids
Polar groups involved in H bonding as donor + acceptor.
- can from phosphate esters
Y, S, T
Sulphur containing a. acids
C - similar to serine, weak H bonds, much stronger acid, forms disulphide bonds
M - fairly hydrophobic, AUG start codon
Acidic a. acids + their amides
Form salt bridges + polar interactions w/ water (H bonding), negatively charged
D & E
- pKa of side chain ~ 4
Amides are N & Q - not ionisable but highly polar, strong H bond donor/acceptor
Basic amino acids
N atoms w/ free electron pair, +ve charges.
R & K
Arg pK =12.5, Lys pK = 10
Peptide bonds
Formation eliminates ionizable carboxyl + amino groups (removes charge)
Repeating N-C-C unit makes up backbone of peptide chain
Secondary structure
A. acid sequence determines regularly repeating conformations.
a -helices + B strands/sheets
Native conformation is single stable shape of polypeptide chain
Affected by bond rotations + weak non-covalent interactions.
Peptide group always planar/flat, C-N bond has double bond character -> resonance
So no rotation at peptide bond, 6 atoms lie in same plane
Trans + cis conformations of peptide groups
Double bond character means distinct conformations possible about C-N.
Cis is less favourable due to steric interference of a-C side chains
Rotation of N-C(a) bind in proline restricted due to ring structure.
Ramachandran plot can model possible phi (N-C(a))+ psi (C(a)-C) rotations
a-helix
Right handed - backbone turns clockwise from N terminus.
- each C=O (n) from H bind w/ amide hydrogen on residue (n+4)
- all C=O groups point towards C terminus, helix is dipole
- stabilised by many H bonds
Pitch is advance along long helical axis per turn (0.54nm)
Rise is the advance of each residue along long helical axis (0.15nm)
-> 3.6 amino acids per turn
B strands/sheets
Strands - polypeptide chains almost fully extended
Sheets - many strands arranged side by side, stabilised by H bonds between adjacent strands (C=O & -NH), if antiparallel more stability.
-> side chains project alternately above + below B sheet plane -> pleated
Loops - contain hydrophilic residues + found on protein surfaces
Turns - loops w/ 5 residues or less
^^ have both B-strands + a-helices
Tertiary structure
Stabilised mainly by non-covalent interactions + disulphide bridge formation.
Hydrophobic residues on isde (entropically favourable)
Determined by X-ray + NMR, sorted patterns into motifs/supersecondary structure + domains
Motifs
At least 2 connected secondary structure elements
- helix-turn-helix, helix-loop-helix
- Coiled coil, long loose coils of 2 proteins w/ hydrophobic interactions
- BaB unit, B strands parallel joined via a-helix
- helix bundle, 3-5 helices
- beta hairpin
- Greek key, mainly B strands
Domains
Independently folded, compact units in proteins, cant be connect via loops
e.g. pyruvate kinase has 3 distinct domains
(often standalone function)
-> illustrate evolutionary conservation of proteins (cyt C highly conserved single domain protein)
Carbohydrate structures
Empirical formula (CH2O)n
Oligosaccharides: 2-20 mon.
Polysaccharides: >20 mon.
Glycoconjugates: linked to proteins/lipids.
2 monosaccharide families: aldoses (=O oxidation at C1), ketoses (=O oxidation at C2)
Glyceraldehyde is smallest triose, asymmetric C2 atom so chiral (enantiomers)
Fischer projection in carbs
Most oxidised C at top (=O), if OH on left (L), if OH on right (D).
-> D isomers more common nature
D/L determined by C furthest away from aldehyde/ketone.
Diastereomers are optical isomers that are not enantiomers.
Hayworth projection
More realistic view of carbohydrate - ring formation.
Pentoses + hexoses are cyclic
Hemiacetal isomeric forms (anomers)
At C1 atom:
- B has OH at C1 on top
- a has OH at C1 on bottom
Sugars w. 6 membered ring (5C+O) - pyranoses (hexoses)
Sugars w/ 5 membered ring (4C+O) - furanoses (pentoses)
Glycosidic bond
Links monosaccharides: anomeric C1 binds OH group (hemiacetal -> acetal)
a1->4 forms bent chain
B1->4 forms straighter chain
Storage of D-glucose
Starch: mixture of amylose + amylopectin
- Amylose is D-gluc in compact helical spiral (a1->4 bonds)
- Amylopectin is D-gluc in branched chain (linked by a1->6 binds), branches every 24-30 glucose units
Glycogen: similar structure to amylopectin but larger + more branches (every 8-12 residues)
-> more compact + faster metabolism
Structural functions of D-glucose
Cellulose: plant cell wall, B1->4 linkages so straight unbranched chains, H bonds w/ adjacent chain to form fibrils -> insoluble polymer
Can also be linked to proteins for recognition, adhesion, secretion.
- O-glycosylation in Ser + Thr residues (-Oh group) forms (a) linkage
- N-glycosylation in Asn residue (-CONH2 group) forms (B) linkage
e.g. elastase is secreted serum glycoprotein w/ linked carbs on surface
Nucleic acids
Monomers has 5C sugar, heterocyclic N base + P group
- sugar can be ribose or deoxyribose (C2 has no OH group)
Deoxynucleotide triphosphates (dNTPs) linked by phosphodiester binds between 3C & 5C
-> pyrophosphate lost from dNTP
- purines (A/G) larger
- pyrimidines (T/C)
2 H bonds between A & T, 3 between G & C
Triacylglycerols
Glycerol backbone w/ 3 fatty acid (acyl) groups attached - hydrophobic.
- important fuel source
- 3 f. acids esterified to glycerol
Glycerophospholipids
Glycerol backbone w/ phosphate moieties, polar head group (can have glycol-conjugates) - amphipathic.
- most abundant lipid in membranes
- 2 f. acids esterified to glycerol
- 1 P esterified to C3 on glycerol, small polar head group pinked to it
Sphingolipids
Built on sphingosine backbone, often has glycol-conjugates - amphipathic.
Ceramide (f. acid chain) added to sphingosine to make it amphipathic.
- sphingomyelin has N & P
- cerebroside has glucose or galactose
- ganglioside has complex oligosaccharide
Isoprenoids
Formed from isoprene (5C block), inc steroids, lipid vitamins, hormones e.g. cholesterol
- largely hydrophobic w/ variable polar group content
e.g. cholesterol is amphipathic w/ small polar head group, component in membranes, precursor to other steroids
-> has fused ring system so less flexible than f. acid chain (fluidity buffer for membranes)
Variation in fatty acids
Differ in hydrocarbon length, most 12-22 long, degree of unsaturation + position of double bonds
- can be monounsaturated (oleate) or polyunsaturated (linoleate)
Cis double bonds introduce kinks - fewer vdw interactions so more fluid (lower melting point)
Biological membrane
25-50% lipid, 50-75% proteins
lipids provide permeability barrier, proteins regulate all other processes
2 leaflets of bilayer differ in lipid composition: outer leaflet has mor sphingolipids, more glycerophospholipids in cytosolic leaflet.
Fluid mosaic model - proteins float in lipid bilayer sea
a) lateral diffusion (in leaflet/monolayer) is very rapid
b) transverse diffusion from one leaflet is very slow
3 classes of membrane proteins
Integral - intrinsic/ transmembrane, contain hydrophobic regions + usually span entire bilayer
Peripheral - proteins associate w/ membrane though charge-charge or H bonds to integral proteins or polar head group of mem. lipids, more readily dissociated from mem (pH or ionic strength)
Lipid anchored proteins tethered to mem via covalent bond
- ester/thioester linking Ser/Cys to fatty acyl group
- amide linking N-terminal Gly to fatty acyl group
- Thioether linking Cys to isoprenoid chain
-> all in cytosolic leaflet
- protein can be anchored to GPI by its C terminus (outer leaflet of mem)
Properties of enzymes
Stereospecific - enzymes usually act upon 1 stereoisomer or substrate
Reaction specificity - product yields essentially 100%
Don’t affect equilibrium itself + remain unchanged.
Simply lower activation energy so speed up attainment of reaction equilibrium
Catalysis + role of active site residues
Substrate specific but not complementary so active site fits better after binding -> induced fit
AS most complementary in shape to transition state, TS stabilisation.
Active site provides binding energy + has catalytic functional groups (nucleophiles, electrophiles, proton donors/acceptors)
- cofactors can provide extra functional groups
Enzyme kinetics
v is proportional to [S]
k (rate constant) is specific to particular process
Catalytic step for ES to E + P is slower rate limiting step (K2), considered irreversible.
Michaelis Menten
Km is enzyme dissociation constant, measure of affinity.
Vmax is velocity when enzyme saturated w/ substrate (high [S])
-> proportional to [E]
Kcat = K2
at saturating [S], Vmax = k2*[E(total)]
higher the Kcat/Km, more efficient the enzyme reaction
Lineweaver-Burk plot
linear transformation of M-M equation
- used to find V0 at saturating [S]
It plots 1/v0 (y) against 1/[S]
-> can obtain kinetic parameters from intercepts
Classification of enzymes
Oxidoreductases (dehydrogenases) - catalyse redox reactions
Transferases - group transfer reactions
Hydrolases - hydrolysis, proteases (chymotrypsin), lipases, esterases.
Lyases - lysis, water not added, leaves double bond e.g. decaroxylases, aldolases, dehydratases
- some lyases, reverse more important (addition to a double bond) -> synthases
Isomerases - isomerism, transfers groups within a mol to give different structural/geometric isomers e.g. isomerases, racemases, epimerases, mutases
Ligases - ligation (joining) of 2 substrates
Enzyme regulation
Key metabolic enzymes show non M-M enzyme kinetics - V0 vs [S] is sigmoidal not hyperbolic.
- they are allosteric, so activity controlled by changing 3D structure due to small mols binding to secondary regulatory site -> conformational change
Also exhibit cooperativity between subunits SO binding by substrate affects subsequent binding at all other subunits -> more active form
Allosteric enzyme forms
Binding activator stabilises active (R) from.
Binding inhibitor stabilises inactive (T) form -> non-functional active site(s)
e.g.phosphofructokinase-1(PFK-1)
Catalyses F-6-P to F1,6-BP in glycolysis.
- ADP is allosteric activator
- PEP is allosteric inhibitor (product of later reaction, example of feedback inhibition)
Plot is sigmoidal due to cooperativity between PFK-1 subunits.
Allosteric regulators affect Km not Vmax
Enzyme inhibition
Competitive - Km is increased , Vmax is same, EI + S
Uncompetitive (rare) - only binds ES complex -> ESI, Km + Vmax decrease, cannot be overcome by more [S], lines on Lineweaver plots parallel, constant Km/Vmax
Non-competitive (rare) - binding affects shape of active site so S can bind but not converted, Km not changed, Vmax reduced, not overcome by more [S], intersect on x axis
Competitive + mixed inhibition common. Mixed affects both Km & Vmax
Homgenisation
1st step in protein purification.
-> physical disruption of cells/ tissues in to membranes, matrixes + organelles.
Can be done w/ mechanical blender, liquid homogenization, sonication (sound waves), freeze thaw cycles, manual grinding
Differential centrifugation
2nd step in protein purification.
-> isolates different cellular components
500g gives nuclear fraction, 10,000g gives mitochondrial fraction, 100,000g gives microsomal fraction.
a = w^2 * r
w is angular velocity (radians/sec OR 2pi rpm/60)
r is radius in m, average is used
RCF = a/9.8 OR rw^2/g
-> doubling speed increases RCF 4 fold
-> doubling radius, doubles RCF
How are proteins separated?
Fractionation based on solubility - competing solutes added (crude separation), precipitated proteins removed by centrifugation. Competing solutes v. water soluble. Cheap + pure.
Dialysis by size - uses permeability of selective membranes. Smaller mols filtered out (passive transport) but large proteins remain
Size exclusion & ion exchange chromatography
Size exclusion chromatography (SEC) - gel has fixed size of pores/beads
- larger mols elute first as they have smaller retention time & cannot enter pores
- smaller mols collected last, longest retention time + travel slowest as can explore all available pathways
Ion exchange chromatography, basic of charge.
- positive charged protein bind negative beads, increasing NaCl conc can increase its elution (competition)
- negative charged protein flows through
Other types of chromatography
Affinity - immobilised molecule w/ affinity used to trap protein of interest in column
Immunoaffinity - antibody against protein of interest
Immobilised ligand - substrate analogue or inhibitor binds enzyme/protein (e.g. heparin)
Lectin based affinity - lectin binds glycosylated proteins, elution is via addition of sugars
Immobilised metal affinity - metal ions (Ni2+) bind engineered recombinant proteins that have poly-his tag at N- or C- terminus.
- ions binds histidine tag
- Ni2+ can bind 6 ligands
Polyacrylamide gel electrophoresis
SDS can convert protein to linear denatured polypeptide complex w/ a negative charge + uniform shape using hydrophobic tail.
Reducing agents need to be used to cleave S-S disulphide bridges to unfold protein.
Proteins move through gel to +ve anode. Gel then stained w/ dye + bands develop.
Abundance: band intensity
Protein purity: separated as bands vertically on a lane
Isoelectric focusing
At pH = pI, protein charge is zero.
Shape of curve is due to property of each protein.
Proteins migrate through gel, charge changes due to change in medium pH
-> accumulate in narrow bands where their charge is zero
MASS spectrometry
Identifies proteins by measuring molecular mass in gas phase.
Protein ionized + fragmented then separated based on mases by magnetic field (m/z, mass/charge ratio)
Ions detected relative abundance vs m/z.
Identifies protein from peptide mass spectrum after proteolysis.
Can display proteomics using protein expression maps
Antibodies in studying proteins
Polyclonal - mixed pop of different antibodies that recognise different epitopes on antigen
Monoclonal - uniform pop of antibodies that recognise same epitope in antigen
For separation: immunoaffinity, immunoprecipitation
For identification: western blotting, immunofluorescence
Western blotting - adds radiolabelled specific antibody to SDS-PAGE polymer sheet, then exposed + developed.
Immunofluorescence - antibody labelled w/ fluorescent mol, use microscope to visualise.
-> actin labelled w/ RFP
-> MTs labelled w/ GFP
-> Nuclei labelled w/ blue dye (DAPI) binds DNA
Misfolding of proteins
Can cause disease:
Mislocalisation proteins: cystic fibrosis, hypercholesterolaemia, cancer
Extracellular toxic aggregates: Alzheimers, Huntington’s.
Abnormal collagen assembly: Marfans syndrome, osteogenesis imperfecta, scurvy
Abnormal cell/tissue morphology:
impaired function -> sickle cell anaemia, cataracts
Exceptions to protein folding rule
Intrinsically disordered proteins can have biological function:
- not completely folded, low complexity
- disordered regions can fold upon binding
- can have flexible linkers
Levinthal paradox: proteins go from unfolded chain to folded chain by conformational search.
BUT takes wayyy too long
SO solution is to fold through semi-stable intermediate states
Thermodynamics protein folding
Spontaneous when change in G is negative, can be driven by decrease in enthalpy or increase in entropy.
Folding protein unfavourable for entropy -> restricts degree of freedom
Water entropy increases as it is displace around unfolded chain.
Driving place for folding is large increase in entropy from release of water previously around hydrophobic groups.
Can polypeptide chains fold unassisted?
Info needed to fold is in primary sequence (Anfinsen)
BUT folding complicated by other proteins in cytosol (non-specific interactions)
Chaperons stabilise intermediates, minimise misfolding + prevent aggregation.
e.g. heat shock proteins (Hsp), named according to molecular weight
Metabolism concepts
Complex mols broken down to release energy - catabolism
Complex mols made from simpler ones to store energy in chemical bonds - anabolism
Multistep pathways -> release of energy in smaller amounts (controlled), each reaction catalyzed by different enzyme.
Mechanisms of regulation
Feedback inhibition - product of a pathway controls rate of its own synthesis, inhibits an earlier step
Feed forward activation - metabolite early in pathway activates enzyme further down pathway
Coupled reactions
Enzyme catalysed reactions can be sum of 2 coupled reactions (exergonic + endergonic)
e.g. glucose phosphorylation by hexokinase
thermodynamically favourable +14kJ/mol (glucose -> glucose-6P) - 32 kJ/mol (ATP->ADP) = -18 kJ/mol
2 domains hexokinase clamp on bound glucose + keep water out of active centre so cannot interfere.
Energy transfer via ATP
Synthesis driven by processes w/ large change in G < 0, it can then be used to drive pathways/ processes w/ change in G > 0
ATP specialised nucleotide .
Adenosine = adenine + ribose
Mg2+ can be added to stabilise triphosphate.
Hydrolysis ATP/ADP (change in G is -32kJ/mol)
Hydrolysis of AMP to adenosine (-14kJ/mol)
Phosphoester joins triphosphate w/ adenosine.
Phosphoanhydride joins phosphate groups.
Coenzymes
NAD & NADP are specialised electron carriers - carried by nicotinamide side of mol.
Oxidising agent accepts electrons (NAD/NADP)
Reducing agent loses electrons (NADH/NADPH)
-> reduced coenzymes produced, conserve energy
NADH is store of electrons, later oxidised by ETC, -220 kJ/mol, energy used to produce ATP
NAD vs NADP
NADP have extra PO4 group used for enzyme recognition.
NAD used catabolic pathways as oxidising agent. NADP used anabolic pathways reducing agent.
Flavin redox cofactor
FAD - flavin adenine dinucleotide
FMN - flavin mononucloetide
^ both reduced to FADH2 & FMNH2.
Reduction has a semi-reduced form (semiquinone) & and a reduced form (hydroquinone)
Glycolysis overview
Glucose -> 2x pyruvate
Pathway is ‘resevoir’ connected by ‘dams’.
- 1, 3, & 10 are irreversible reactions (points of regulation + control)
1 & 3 are phosphorylation steps:
1. Hexokinase forms Glucose-6-P + ADP
3. PFK forms fructose 1,6-bisphosphate & ADP
- Pyruvate kinase converts phosphoenolpyruvate -> pyruvate + ATP (sub-level phosphorylation)
Net: 2 pyruvate, 2 ATP, 2 NADH
Hexokinase regulation
G6P inhibits hexokinase (feedback inhibition).
G6P levels increase when downstream glycolysis inhibited -> accumulates.
Glucokinase does same reaction in liver, not inhibited by G6P
Phosphofructokinase-1 (PFK-1) regulation
Most important reg enzyme in glycolysis - allosteric enzyme regulated allosterically.
- ATP, citrate (liver) are inhibitors
- AMP, fructose-2,6-bisP (liver) are activators
AMP can relieve ATP inhibition.
-> increases/decreases rates in response to energy requirements of cell
Other regulators:
- frutose-2, 6-biphosphate important activator eukaryotes (liver)
- high citrate levels inhibitory (biosynthetic precursors abundant)
- high H+ inhibits, pH falls when muscle work anaerobically, so inhibition prevents lactic acid build up
Pyruvate kinase regulation
ATP inhibits its action via covalent modification (phosphorylation via PKA)
F1,6BP has activating effect (feed-forward activation), pyruvate kinase dephosphorylated via phosphoprotein phosphatase I
Link reaction & PDC
Pyruvate decarboxylated by PDC (3C -> 2C) into acetyl CoA.
CoA-SH + NAD+ used, NADH + CO2 formed
acetyl- CoA has higher energy thioester bond.
change in G = -33.4 kJ/mol in link reaction
PDC is large, 3 subunits:
24x E1 - pyruvate oxidative carboxylation
24x E2 - transfers acetyl group -> CoA
12x E3 - cofactor regeneration
It is located inside mt matrix. Pyruvate translocase is H+ symporter which moves pyruvate through inner mem.
Mitochondrial shuttles
Malate aspartate shuttle- uses antiporter, uses oxaloacetate reduced by NADH to form malate, carry e into matrix, then reduce NAD -> NADH.
- Oxoaloacetate converted to a-Ketoglutarate by glutamate.
- a-Ketoglutarate + aspartate can return to cytoplasm via antiporter
Glycerol phosphate shuttle consists of brown dispose tissue.
Regulation of PDC
Irreversible step - commits C to CO2 or incorporation into lipid.
Feedback inhibition - high acetyl CoA inhibits E2, high NADH inhibits E3/high NAD activates.
E3 allosterically activated by F-1,6-BP
Reversible phosphorylation of PDC by PDK (pyruvate dehydrogenase kinase) inactivates enzyme.
Complex I
NADH-ubiquinone oxidoreductase.
Largest w/ 40 subunits, site of NADH oxidation -> NAD+.
Occurs in stages, 4H translocated per 2e transferred.
Crystal structure: Q module connects it w/ membrane, mem arm P module pumps H+, dehydrogenase N-module oxidises NADH (sits in matrix)
Complex II
Succinate-Q oxidoreductase (a.k.a. succinate dehydrogenase in Krebs)
Common to citric acid cycle + inner membrane. Does not contribute to H+ gradient.
Supplies electrons from succinate via FADH2 & Fe-S to ubiquinone (CoQ), reduced to ubiquinol (CoQH2)
- forms fumarate (dehydrogenation)
Complex III + cytochrome C structure
Ubiquinol-cytochrome C oxidoreductase (cyt C reductase)
Transfers e’s from QH2 to cytochrome C .
4H+ translocated to intermembrane space (2 from matrix, 2 from QH2)
Electrons transferred to 2 mols in cytC
-> Fe3+ in cyt C reduced in both stages
Cytochrome C - electron carrier between complex III + IV.
a-helical haem protein, Fe2/3+ at centre does not bind O2.
- small + highly soluble from intermembrane space, associates w/ inner mt membrane
Complex IV
Cytochrome C oxidase.
Receives e’s from cytC (1 at a time).
Fe2 & Cu both reduced + oxidised as e’s flow to oxygen,
Catalyses reduction of O2 -> 2H2O (uses 4H+ from matrix)
Dimer, free energy accumulated used to translocate 2 H+ from matrix -> intermembrane space
Complex V
ATP synthase, 2 subunits F0 & F1.
Proton gradient energy used to synthesise ATP (3H+ needed for 1 ATP)
F1 (knob) has catalytic subunits, F0 (stalk) is the protein channel.
F1 has 3a, 3B, 1y, 1d, 1e subunit. a and B alternate in hexamer ring.
y main part of central axle (rotates inside hexamer ring).
d in peripheral stalk (fixes ring still)
B units have active site for TAP synthesis.
~3H+ per ATP produced + 1 H+ for P transport in via ANT
Rotational catalysis in ATP synthase
B subunits exists as:
open (O) - binds ADP + P
loose (L) - active site closes loosely on ADP + P
tight (T) - ADP+P -> ATP
Proton flow drives F0/ye rotation -> cyclic conformational changes in each B subunit.
Evidence for rotation provided by actin filament when attached to ATP hydrolysis enzyme, ATP provided.
Adenine nucleotide transportase (ANT)
Coupled exchange of ATP(out) + ADP(in) via antiporter.
P then enters matrix in symport mechanism w/ H+.
Photosystem II
> 20 subunit transmembrane assembly, responds to wavelengths < 680nm.
Antenna pigments capture light + transfer it between themselves until it reaches special pair of chlorophyll a mols (P680) in reaction centre - energy trap. Pair oriented in way that they are ionised (1 +ve , 1 -ve).
- e’s transferred to pheophytin -> plastoquinone (PQ)
- 2nd e reduces mobile PQ -> PQH2
- O2 formed as ionised P680+ extracts H20 from manganese centre
electron excited + releases energy to excite electron neighbouring pigment (resonance energy transfer) -> photoinduced charge separation.
Oxygen evolving centre
1 Ca2+ manganese ions, Mn changes its oxidation state so useful for e transfer.
Tyr(Y) residues mediate e transfer centre -> chlorophyll
- has oxygen, calcium + manganese cluster (oxidised 1 e at a time)
water mols bound to Ca + Mn4 linked to form O2 (released from centre
Electron transfer to PQ then to Cyt bf in photosynthesis
To PQ:
- PQH2 diffuses through mem carrying 2e
- 4H+ needed to generate 1x O2, 4 e transferred
- 4H+ from H2O released to thylakoid lumen, 4 H+ from stroma transferred to PQH2
To cyt bf:
- PQH2 oxidised (loses 2e), 1 e at a time via Q cycle
- e transferred to next carrier, plastocyanin (PC)
- 4H+ transferred to thylakoid lumen, 2 from strom 2 from PQH2 (Q cycle)
Photosystem I
Large transmembrane assembly (> 100 cofactors), core made of psaA & psaB.
Special pair P700 absorbs photons. Then transfers e’s to chlorophyll A -> phylloquinone -> 3 iron-sulphur clusters (intermediate)
E’s then transferred to ferredoxin (2Fe-2S cluster). Ionised P700+ recovers e from PC.
NADP reduced to NADPH, ferredoxin oxidised. Catalysed by Fd-NADP+ reductase via semiquinone (FADH intermediate)
ATP synthesis in photosynthesis
Protons diffuse through chloroplast ATP synthase.
Has knob (CF1) and stalk (CF0) structure:
CF0 spans membrane, forms H+ channel for passive transport into stroma, has 12 subunits (12 protons per rotation)
CF1 protrudes into stroma, has catalytic subunits for ATP synthesis
Cyclic phosphorylation
Alternative pathway when no CO2 or NADP so cant accept e from FD.
Net result: 8H+ to lumen per 4 photons
Calvin cycle: fixation
Powered by ATP + NADPH. Converts CO2 into carbs (more reduced). Takes place in stroma.
Fixation of atmospheric CO2 by RuBisCo converts rib1,5-biphosphate (5C) -> 2x 3-phosphoglycerate (3PG)
RuBisCo very inefficient, slow + poorly selective, fixes 3CO2, 16 subunits, can bind O2 instead of CO2 (waste of energy)
Calvin cycle: reduction
Reduction of 3PG -> 1,3 biphosphoglycerate using 2 ATP.
1,3-BPG reduced to glyceraldehyde 3 phosphate by
2 NADPH.
Regeneration of rib 1,5-biphosphate from 2xG3P uses ATP, so more CO2 can be fixed.
1 C used for hexose formation
Calvin cycle: regeneration
Regeneration of rib 1,5-biphosphate from 2xG3P uses ATP, so more CO2 can be fixed.
For each 3 turns: 9 ATP + 6 NADPH used
- 5/6 G3P used to regenerate 3x R1,5BP, so net 1 G3P every 3 cycles
Glucose storage in plants
1 glucose mol needs 6CO2, 18ATP/12NADPH.
Glucose/fructose converted into sucrose, starch, cellulose.
Enzymes used gluconeogenesis
Instead of:
1) Hexokinase -> glucose 6-phosphatase
3) PFK-1 -> fructose 1,6 biphosphatase
10) pyruvate kinase -> pyruvate carboxylase (pyruvate -> oxaloacetate), phosphoenolpyruvate carboxykinase (oxaloacetate -> phosphoenolpyruvate
Takes 6ATP/GTP, more than glycolysis produces (2ATP)
pyruvate carboxylase
tetramer 4 subunits, each has 4 domains
Important at start gluconeogenesis, catalyses irreversible reaction in mt matrix
Has prosthetic group - biotin, can be allosterically activated by acetyl CoA (signals abundant energy)
PC has 2x biotin carboxylase domains + 2x pyruvate carboxylase domains
Pyruvate binds covalently via Lys side chain.
Glycogen as glucose storage + its removal
Stored in cytosolic granules in liver + muscle cells. Dehraded in glycogenolysis.
Branched polysaccharide w/ central glycogenin protein - primer for glycogen synthesis.
Glycogen phosphorylase catalyses sequential removal of glucose residues.
Phosphoglucomutase converts G1P -> G6P
Transferase shifts 3 glucoses from outer branch to another.
Debranching enzyme a-1, 6 glucosidase removes branched glucose - leaves elongated unbranched chain
Glycogen synthesis
Glucokinase in liver converts glucose -> G6P
- has 50-fold higher Km than hexokinase
UTP~ATP
G6P -> G1P -> UDP-glucose (using UDP-glucose pyrophosphorylase)
Glycogen synthase converts UDP-glucose to glycogen.
Linkage is 1,4-glycosidic
-> synthase needs 4 glucose mols already on chain
Glucose transporters
Family of passive hexose transporters, facilitate glucose transport.
GLUT1/3 - nearly all mammalian cells, constant glucose transport under normal conditions.
GLUT2 - liver + pancreatic B cells, transport when glucose conc in blood is high, pancreas detects + insulin produced
GLUT4 - muscle + fat cells, increase rapidly in presence of insulin which binds tyrosine kinase
-> insulin is a 2 polypeptide chain hormone linked by disulphide bonds
Glycolysis + gluconeogenesis regulation
If glucose abundant, glycolysis predominates (vise-versa).
F2,6BP is potent activator of PFK-1 & inhibits F1,6-biphosphatase.
-> produced by phosphorylating F6P by PFK-2 in liver, can be converted back by FBPase2
-> both enzymes controlled by phosphorylation of single Ser29 residue
Also regulated by:
Insulin - increases rate of glucose transport in muscle, adipose tissue, stimulates glycogen synthesis in liver
Glucagon - from a-cells of pancreas in low blood glucose, stimulates glycogen degradation
Epinephrine - produced by adrenal glands due to sudden energy requirement -> stimulates glycogen degradation
Glucagon & liver glycolysis
Low blood glucose - glucagon rises -> phosphorylation of PFK-2/ FBPase2 rises
F2, 6BP decreases SO gluconeogenesis dominates
High blood glucose - glucagon falls + insulin rises -> PFK-2 rises/ FBPase2 loses phosphoryl group
F2,6BP increases SO glycolysis dominates
Amino acid metabolism
Nitrogenase converts N2 -> NH3, needs ATP + reducing power
- tetramer of 2a & 2B subunits
- contains iron-molybdenum (Fe-Mo)
humans have 4/50 Mo containing enzymes e.g. xanthine oxidase
FeMoco is primary cofactor of nitrogenase, site of N2 binding + reduction
NH4+ incorporated into Glu (main chain) & Gln (side chain)
1) a-Ketoglutarate can incorporate NH4+ + forms L-glutamate
2) Glutamate incorporates NH4+ into glutamine (glutamine synthetase)
Transaminase reactions
In a. acid catabolism, transfer amino group -> a-ketoglutarate
Transaminases measure of liver function
Humans can only make 11 a. acids vs most microorganisms can make all 20
Amino acid degradation
In terrestrial vertebrates: ammonia converted to urea (excreted by kidneys)
1) Transaminases, a-amino group transferred to aKG -> glutamate
2) Glutamate dehydrogenase oxidises glutamate + removes NH4+ regenerating aKG
-> reduces NAD -> NADH
Glycerol metabolism
Converted to DHAP in liver, then isomerised to G3P (intermediate in glycolysis pathway)
Fatty acid metabolism
F. acids transported to cells via blood bound albunim.
Enter cells via FATP + bind FABP.
Then oxidised in mt matrix:
- activated on outer mem by acyl CoA synthetase -> fatty acyl CoA (has thioester bond).
- fatty acyl CoA translocated acroos inner mt mem by acyl carnitine translocase (trsnfers acyl group)
- B-oxidation reaction converts 2Cs fatty acyl CoA into acetyl CoA
Fatty acid synthesis
If glucose plentiful, acetyl CoA used in f. acid synthesis in cytoplasm of liver cells +.adipocytes
NADH used, 1st irreversible step by acetyl CoA carboxylase.
Chain elongated by sequential addition of 2C units , 2 NADH each step, fatty acid synthase complex used.
-> esterified by glycerols, stored in adipocytes
