Biochemistry 1 Flashcards
What are the electron orbitals?
s = 2
p = 6
d = 10
f = 14
How many electrons in each shell?
1st = 2
2nd = 8
3rd = 18
4th = 32
What is the shape of the 2p orbitals?
2pz = /
2py = l
2px = –
x then y then z
What is the nodal plane?
The region around a nucleus which the probability of finding an electron is zero.
What is the electron octet?
Noble gas configuration.
Full outer shell.
E.g. F gains an electron from Na - both will have electron octet
What are waves?
Electrons can be presented as waves - in phase = same wave.
What is a covalent bond?
2 electrons shared in 2 overlapping orbitals from 2 atoms with orbitals of similar energy.
What happens when 2 atomic orbitals combine in phase?
They form a bonding orbital which is lower than the original orbitals = bonding molecular orbital.
What happens when 2 atomic orbitals combine out of phase?
They form an antibonding molecular orbital which is higher in energy.
What is a sigma bond?
Strongest covalent bond.
Formed when 2 orbitals (s.p or hybrid) overlap along a line between the two nuclei.
Allows for free rotation around axis.
What is a pi bond?
Formed when two p orbitals overlap laterally - NOT ON BOND AXIS.
Usually occurs in addition to sigma - in double/triple.
Restrict rotation and weaker.
What is a hybrid orbital?
An orbital formed by combination of two or more atomic orbitals.
What is sp3?
Orbital formed from one s orbital and 3 p orbitals from the same atom - this forms four equivalent sp3 hybrid orbitals = tetrahedral (109.5).
Unsymmetrical - one lobe bigger than other.
What are the bonds in methane?
Sigma bond - 1s orbital from H overlaps with sp3 of C
What are examples of sp3 hybridized atoms?
Oxygen in water
Nitrogen in ammonia
1s of H overlaps the sp3 orbital, some sp3 orbitals have lone pairs
What is a molecular orbital?
Combination of atomic orbitals of similar energy - can be bonding or antibonding
What does in and out of phase mean?
In = electron waves are the same
Out = electron waves are different
What is an ionic bond?
Electron transferred - orbitals far apart in energy.
What does the excited state mean?
Ground - normal
Excited - moves to higher energy level e.g. s electrons move to p = hybridisation to sp3
Why can oxygen bind to 2 things?
The electrons are excited to sp3 orbitals - two have 2 electrons, 2 have 1 electron so can only make 2 covalent bonds.
How is the c-c bond formed in ethane?
Overlap of 2 carbon sp3 orbitals = sigma
What is an sp2 orbital?
Trigonal arrangement
1 s blends with 2 p
What is the bonding in ethene?
Sigma bond between c-c (sp2-sp2)
Sigma bond between c-h (sp2-s)
pi bond between c-c (p-p)
What is the bonding ethyne?
Sigma bond between c-c (sp-sp)
Sigma bond between c-h (sp-s)
2 pi bonds between c-c (py-py and pz-pz)
Triple bond
What are the bond angles?
single c-c = 109.6
double c-c = 121.7
triple c-c = 180
What is non-polar covalent bonding?
Equal sharing of electrons
What is polar covalent bonding?
Sharing electrons between atoms of different electronegativities.
What is the difference between sp2 vs sp3?
sp2 = one s and two p = 3 sp2
sp3 = one s and three p = 4 sp3
What does the A with a circle on top mean?
0.1 nm
Angstrum
What are the shapes of sp orbitals?
sp = linear
sp2 = trigonal
sp3 = tetrahedral
What effect does s character have?
More character - shorter, stronger and larger bond angle
What aa’s are proteins made of?
L amino acids (d is an isomer but not used - but just as good)
What types of side chains do amino acids have?
Non polar
Polar
Polar positively charged
Polar negatively charged
What is a disulfide bond?
Between two cysteines to make a cystine
What direction do polypeptides go?
Written from N to C terminus
What are the features of a peptide bond?
Very stable and planar (can’t move due to partial double bond character)
Partial double character
Cleaved by proteolytic enzymes
c–o = sp2
What rotation is in amino acids?
N-C = phi (line in circle)
C-C = psi (trident)
What did Ramachandran say?
Many combinations of phi and psi are not found because of steric clashes
What are the bond lengths?
Single = 1.54 A
Double = 1.33 A
Triple = 1.20 A
Why can’t the peptide bond rotate?
Due to the delocalisation of electrons from the double-bonded oxygen to the peptide bond.
What is the denaturation of ribonuclease A?
It is reversible.
Native catalytically active state + urea & beta mercaptoethanol –> unfolded inactive with disulfides reduced to Cys –> removal of urea & mercaptoethanol = restored.
This showed that the instructions to fold in in the protein sequence.
What is the free energy required to denature AA’s?
0.4 kJ/mol per Amino Acid
What free energy is needed to overcome hydrogen bonds?
12 kJ/mol
What group does cysteine have?
SH = thiol
What covalent bond do adjacent cysteines make?
A disulphide bond s-s
Requires oxidative conditions
Only bond formed between side chains
Can provide extra stability
What are the non-covalent forces which hold proteins together?
- ionic interaction
- van der waal interactions
- hydrogen bond
- hydrophobic effect
What is the energy of association?
E = k q1 q2 /Dr
q1 & 2 = electric charges
r = distance
k = 9 x 10^9 JmC-2
D = dielectric constant
What is one electrical charge?
1.6 x 10^-19 C
What is D?
Dielectric constant of a solvent.
Is a measure of its ability to keep opposite charges apart.
Vacuum = 1
Water (polar) = 80
Non-polar (interior of protein) = 4
What are the three wan der Waal interactions?
Dipole-dipole interactions
Dipole-induced dipole interactions
London dispersion forces
What is a dipole-dipole interaction?
Occur between polar molecules which have permanent dipoles.
e.g. between c–o, c–o
What is a dipole-induced dipole interaction?
Between polar and non-polar (creates temporary dipole)
e.g. c–o, h3c
What is a london dispersion force?
Present in all molecules due to fluctuating asymmetric distribution of electrons.
e.g. ch3, ch3
What is a hydrogen bond?
Interaction between polar groups (H and FON)
Partial negative on O/N/F - partially positive on H.
Strongest non-covalent in aqueous medium
Strongest tend to be linear with lone pair orbital
What is the energy association of non-covalent interactions?
Hydrogen bonding = 4-13 kJ/mol
Ionic interactions = 5 kJ/mol
Van der waal = 2 kJ/mol
Which is longer: covalent or hydrogen bonding?
Hydrogen
What is the hydrophobic effect?
Influences which cause nonpolar substances to minimise their interaction with water - form micelles in aqueous solutions.
Non-polar
What can water solubilise?
Polar, ionic and hydrophilic
How does water act with hydrophobic parts?
Forms ‘cages’.
When buried - caged water molecules are released which increases entropy.
How do proteins fold?
Spontaneous - gibbs needs to be negative.
Enthalpy change is slightly negative.
Entropy change is positive as caged water is released.
What is the gibbs equation?
^g = ^h - t^s
gibbs free energy = enthalpy change - T entropy change
negative = feasible
Which non-covalent interactions stabilise proteins?
Hydrogen bonds = strong
Ionic = strong but don’t stabilise well
Dipole-dipole = weak but stabilise well
Why do proteins need to fold?
Residues need to come close to eachother to help function
NEED TO KNOW ALL PROTEINS?
What is the primary structure of a protein?
Linear sequence on amino acids
From N terminus to C terminus
What is the secondary structure of a protein?
Stabilised by hydrogen bonding
- alpha helix and beta pleated sheet
What is an alpha helix?
CO and NH hydrogen bonded - every 4 aa’s
1.5 angstrum rise per aa
100 degree rotation
3.6 aa per turn
RIGHT HANDED
Dipoles of each peptide bond align
What is the alpha helix terminator?
Pro (proline)
Are alpha helices hydrophobic or hydrophilic?
Amphiphilic - have both
Helix has a hydrophobic and hydrophilic sides
Occur in globular proteins - hydrophobic face interior, hydrophilic face solvent.
What is a beta sheet?
Can be parallel and antiparallel
Side chains occur on opposite faces of the sheet.
Can be flat - sometimes twisted due to steric repulsion.
Can have a beta turn.
What is the supersecondary structure of a protein?
Combination of secondary structures (e.g. beta-alpha-beta, alpha-alpha)
What is the tertiary structure?
Assembly of secondary elements into native protein structure
What is the quaternary structure of a protein?
Multiple polypeptide chains assemblied
homooligomer - identical monomers
heterooligomer - different
What is an allosteric interaction?
Ligand binds to quaternary structure - alter affinity of ligand to another subunit
What are domains?
Independent units for polypeptides >200 aas - binding sites in clefts between domains
e.g. DNA polymerase - polymerase domain (synthesises new DNA), exonuclease domain (degrades incorrect DNA)
What is an immunoglobulin?
Each domain in IgG is similar - antiparallel beta sheets surrounding hydrophobic core
What is a conformative change?
Many proteins change their shape when binding to a ligand (on active site) - non-covalent bonds form
e.g. lactoferrin changes shape to show when it is bound to iron.
What is phophorylation?
OH of ser, thr and Tyr can be reversibly phosphorylated
What is glycosylation?
Addition of carbohydrates - increases hydrophilicity and ability to interact with other molecules.
On asn or can be ser and thr
What is hydroxyproline?
OH group added to proline - this stabilises collagen fibres - Vit C deficiency can inhibit this
What is y-carboxyglutamate?
Vitamin K deficiency can result in low carboxylation of glutamate in prothrombin - can cause haemorrhage.
What are protein families?
AAs with a closely related amino acid sequence - arises from common ancestor
What are serine proteases?
Family of enzymes which all contain - asp-his-ser
Includes: chymotrypsin, trypsin (arg) and elastase (val and thr)
Have very similar structures and include digestive enzymes and blood clotting
What is a reaction mechanism?
1) determine where the electrons are
2) determine what bonds are made/broken
3) describe flow of electrons
What is a lewis structure?
1) draw molecular skeleton
2) assume bonds are covalent
3) count electrons
4) add sigma bonds
5) add pi if necessary
What is an electro and nucleophile?
Electrophile - accepts electrons
Nucleophile - supplies electrons
How would we draw a sigma bond being made?
e.g. when an anion and cation encounter
Arrow is from lone pair of electrons to where they move to.
Reversible
What is substitution nucleophilic bimolecular?
A nucleophile attacks an electrophile - substitutes a different group which is opposite to the attack.
There’s a single transition state where they are both partially bonded
e.g. bromine replacing iodine
What is a lysozyme?
Enzyme - first line of defense as cleaves peptidoglycan (in cell walls of gram-positive bacteria - no effect on gram negative)
Part of glycosidase enzyme group
Hen egg-white lysozyme was first crytallography of any enzyme
How do lysozymes work?
Cuts a glycosidic bond between NAM and NAG sugars (have similar structure) in peptidoglycan
Glycosidic bond between 4th and 5th sugars break
What is the structure of a lysozyme?
129 aa’s - four disulphide bridges
Has 2 domains seperated by deep cleft (active site and can bind to 6 sugars- ABCDEF)
L - beta sheet with hydrophilic residues
R - hydrophobic core surrounded by a-helices
What is the lysosyme active site?
Binds to peptidoglycan so a NAM ring is at D and NAG at E.
The D-E bond is next to Glu35 and Asp52 - Glu35 has a carboxylic acid chain (as its in unusual hydrophobic environment) and Asp52 has a carboxylate at pH 6 (optimum)
What is the lysozyme mechanism?
1) Nucleophilic attack from ASP 52 - forms covalent acyl-enzyme intermediate
2) Glu35 donates proton and sugars E, F diffuse away
3) Water now attackes - OH to D and proton to Glu35 - ABCD released
DRAW MECHANISM
What are the products of a lysozyme reaction
Sugars E,F
Sugars ABCD
What do enzymes do?
Enhance rate - don’t alter equilibrium
Have active sites - for their job
Unchanged by end of cycle - may have to use ions from water ect. to get back to normal
What is the difference between pH, Ka and pKa?
pH = conc of hydrogen ions
pKa = strength of acid
ka = dissociation constant
What is the dissociation constant?
Ka = [H][A]/[HA]
What is pKa?
pKa = - log(Ka)
pKa basically turns Ka into a nice number
What is the Henderson-Hasselbalch equation?
pH = pKa + log([A]/[HA])
We can use it to work out the % of protonated and deprotonated forms of a group.
pKa>pH = protonated vice versa
e.g. Histidine side chain pKa = 6, so at pH 7
log([A]/[HA]) = 1
[A]/[HA] = 10
for every histidine sidechain in HA form, there’s 10 in A- form
10/11 = 91% of sidechains deprotonated
How does pH change as hydroxide is added?
pH increases - cation (nh3+) then zwitterion then anion (coo-)
Some plateaus in graph - pK1, pl (around 6-9), pK2
What is pl?
Isoelectric point - zwitterion
How many pKa values in an amino acid?
2 or 3 if the side chain is titratable
Amino group - pKa = 9-10
Carboxyl group - pKa = 2-3
What effect does the side chain of histadine have on pKa?
The chain has a pKa of 6 - makes it very sensitive to pH under normal conditions
Are most enzyme reactions pH sensitive?
e.g. Cholinesterase increases and plateaus
e.g. Chymotrypsin normal bell curves
e.g. pepsin is high at low then decreases to 5
The protonation state of side chains are usually responsible for this
Why can the local environment influence sidechain pKa?
In lysozymes - Glu35 has a pKa is 4.1 but the pH that it usually works at is 7.4 - expect it to be unprotonated - BUT IT’S NOT
What is the name of a protonated and unprotonated form of the histidine side chain?
Protonated = imidazolium ion
Unprotonated = Imidazole
What groups do papain have?
Cys25 - wants to be unprotonated - 4.2
His15 - wants to be protonated - 8.2
Has bell-shaped curve
How does an enzyme make a reaction faster?
Turns a intermolecular reaction into a faster intramolecular one.
Holds them in an optimum orientation so they have react.
This is rate enhancement
Are there intermediates from substrate to product?
Yes!
There may be intermediates (lower curve) or transition states (high curves) - intermediates are more stable.
Enzymes prefer to bind transition states
How do enzymes bind to substrates?
Increase rate through proximity and orientation effects
They bind to transition state.
They do not bind too strongly (hypothetically great) as that would increase the activation energy to the non enzyme levels
E+S - ES - ES’
How does the induced fit theory work?
Substrate binds non-optimally and is stressed when bound - enzyme strained but the strain energy is released when transition state is reached
What is catalysis?
The process that increases the rate at which a reaction approaches equilibrium
What steps occur in enzyme catalyzed reactions?
1) general acid-base catalysis - donation/gain of proton
2) covalent catalysis
3) metal ions in catalysis
Lower free energy pathway
What is an example of acid-base catalysis?
Lysozyme - Glu35 donates a proton to the substrate and cleaves the glycosidic bond - acts as an acid. Glu35 then takes a proton from water - acts as a base, OH from water adds to substrate - complete cycle
What is Vmax and km?
Vmax = maximum velocity (rate)
Km = 1/2 Vmax
What are enzyme inhibitors usually?
Transition state analogs
What is covalent catalysis?
Some enzymes can form covalent bonds with substrates - generate impermanent intermediates
Usually involves a strong nucleophile on enzyme
How does a lysozyme use covalent catalysis?
Forms an intermediate between Asp52 and the oxonium ion on NAM - a water will donate oxygen to break bond - don’t worry about details for exam
What metal ions are used in catalysis?
Good as don’t alter pH
Metal can be tightly bound - metalloenzymes
e.g. Fe2+/3+, Cu2+, Zn2+, Mn2+
Or loosely bound - metal activated
e.g. alkali earth metals - Na+, K+, Ca2+, Mg 2+
Metals have coordination shells
How are metals used in catalysis?
- can generate nucleophilic species to participate in reaction e.g. carbonic anhydrase uses Zn to generate OH- nucleophile to attack CO2
- can stabilise transition state charge e.g. DNA polymerase I has Mg2+ or Mn2+ bound to active site which stabilises phosphate transtition state
- can increase binding interaction e.g. Mg2+ can bind to ATP and indirectly to the enzyme via water
- can use oxidation state to facilitate catalysis e.g. Fe-S clusters can change from Fe2+ and Fe3+
What are cofactors?
Additional metal ions or small molecules which help enzymes with their function.
- usually recycled and used again
Usually are coenzymes (small organic)
- tightly bound (prosthetic group)
- loose or dissociate (cosubstrate)
What are coenzymes?
Small organic cofactors
can be tightly bound (prosthetic group)
can be loose or can dissociate (cosubstrate)
What are the cofactor + enzyme called?
Enzyme alone = apoenzyme
Together = holoenzyme
Can proteins help enzymes?
Yes!
They are a protein coenzyme and usually involved in transport
What is AMP?
Adenosine monophosphate
- building block in cofactors
- phophate group, adenine ring (2) and ribose ring
What are nucleoside triphosphates?
ATP, GTP (guanine) ect.
- know structure
What is NAD?
Nicotinamide adenine dinucleotide
- nicotinamide ring, adenine ring, 2 ribose, 2/3 phosphates
- cofactor
- can be NAD+/NADH/NADP+/NADPH
- the nicotinamide ring is what gets reduced
- Used to accept or donate hydride group
- need to know this structure (especially nicotinamide)!!!
What are the different classes of enzymes?
Oxidoreductases - redox
Transferases - transfer things
Hydrolases - transfer things involving water
Lyases - make double bond
Isomerases - intramolecular group transfer
Ligases - joining molecules by ATP
What are oxidoreductases?
Often use a cofactor e.g. NAD
Carries out REDOX
Half equations can show this
What are transferases?
Transfers a group between molecules e.g. nucleophilic substitution
Group transfer reactions - transfer of an electrophile from one nucleophile to another
What is a group transfer reaction?
Transfer of electrophiles from one nucleophile to another
What is a hydrolase enzyme?
Cleavage reaction via addition of water
e.g. aryl sulphatase
What is a lyase enzyme?
Also known as synthases
Add or remove groups to make double bond
e.g. enolase
What is an isomerase enzyme?
Interconversion of isomeric forms of compounds
When converting L-AA to D-AA - racemization
What is a ligase enzyme?
Joining two molecules requiring ATP
e.g. CO2 + pyruvate –> oxaloacetate needs pyruvate carboxylase - requires biotin cofactor
What is a membranes permeability?
Permeable: gases, ethanol
Slightly permeable: water, urea (small uncharged polar)
Permeable: glucose, ions, charged polar molecules (AA, ATP)
What fats are in membranes?
Phospholipids - glycerol + 2 FAs + phosphate attached to alcohol
- phosphatidyl serine
- phosphotidyl choline
- phosphotidyl ethanolamine
- phosphotidyl inositol
Know structure??
Can influence fluidity, curvature and thickness
How does membrane composition change between cell types?
RBCs - equal amounts of cholesterol and the phospholipids
Neurones - more P-ethanolamine and P-serine and cholesterol
E.coli - only P-ethanolamine and P-serine
Endoplasmic reticulum - more P-choline
Mitochondria - more P-serine, P-ethanolamine, P-choline
don’t need to know well
What does cholesterol do to membranes?
Makes them thicker as it has a lipid-ordering effect
Has hydrophilic (OH at end) and phobic parts (steroid rings)
What causes membrane curvature?
Segregation of lipids - depends on size of heads and tails
P-choline - cylindrical lipids - bilayers flat
P-ethanolamine - cone shaped - curved
What determines membrane fluidity?
Fatty acid composition and cholesterol
Cholesterol - stiffens the structure and makes thicker
How much proteins do membranes have?
25-50% lipid and 50-75% protein by mass
Many membrane proteins diffuse rapidly in the plane - fluid mosaic model is used for the organisation of everything
What is a leaflet?
Half of a phospholipid bilayer
How can we visualise the movements of proteins in the bilayer?
Fluorescence recovery after photobleaching
1) cells are labelled with a fluorescent reagent binding with specific lipid or protein
2) laser light is focused on a small area - reduces fluorescence and bleaches
3) the fluorescence will increase as unbleached surface molecules diffuse into it
How do proteins provide functional asymmetry of membranes?
Inserted asymmetrically e.g. Na+/K+ pump is orientates so Na comes out and K+ goes in.
The asymmetry is preserved as proteins can’t rotate - just move side to side
What are the three types of membrane proteins?
- integral (intrinsic)
- peripheral (extrinsic)
- lipid anchored
What are integral membrane proteins?
All or partially embedded in membrane - require detergent to release them
Often transmembrane
Often use alpha helices to span membrane
Can use beta sheets (8-22) wrapped around to form beta barrels - usually pores or receptors
Interact with hydrophobic interior
What are peripheral membrane proteins?
Interact with membrane via polar heads of integral proteins
What are lipid-anchored membrane proteins?
Protein polypeptide remains in aqueous phase
Can be fatty acid anchored, isoprenoid anchored or glycosylphosphatidyl-inositol anchored
What are the two classes of membrane transfer proteins?
Channels/pores - central passage for ions/molecules
Transporters - passive and active
What are the transporters transfer process?
Uniport - single solute
Symport - same direction
Antiport - opposite direction
What are the two types of active transport?
Primary - coupled to energy source
Secondary - coupled to ion conc gradient created by primary
What is ModABC?
ATP-binding cassette transporter
What is the work equation?
work (J) = force x distance
e.g. dropping apple from string
Starts at rest then moves in the direction needed to reduce potential energy
Lowest part of the graph is where the particle would be
What is pico?
x 10-12
e.g. piconeutons per nanometre
What is kinetic energy?
KE = 1/2mv2
Energy is conserved
If a ball bangs into another - kinetic energy remains the same before and after
What is the Boltzmann distribution?
P directly proportional e^-PE/kbT
P = probability of energy
e = exponential
PE = potential energy
kb = boltzman constant
T = temp (kelvin)
We can use this equation to find the difference in probability of energies
Higher probability when energy is lowest
What is KbT?
Typically the energy of one molecule
Boltzmann constant (Kb) (JK-1) x T (temp)
Units = J
To find energy per moles - do RT (gas constant)
Is PE the same from folded to unfolded?
Yes!
e.g. if increased by 10 when folded, decreased by 10 when unfolded
Change of potential energy = change of enthalpy
Does an unfolded or folded protein have more entropy?
Unfolded - at higher temperatures get more probable - still not as likely as folded
How is free energy useful?
Allows coupling - favourable can drive unfavourable e.g. ATP synthase uses H+ concentration to try to increase ATP when the conditions would want to decrease it. Overall delta g = delta g of H transfer + delta g of ADP-ATP reaction
Overall delta G must be negative enough for reactions to occur
Delta g for H would be negative, delta g for making ATP is positive - add together should be negative
What is the entropy of mixing?
If we shake 2 solutions together of the same size and shaped particles - all microstates equally likely
Small number of states = low entropy = high free energy vice versa
More favourable to be mixed than ordered
When we mix - more microstates so lower g
What is the free energy for transferring one molecule from a to b?
delta G = kb x T x ln (Cb/Ca)
in moles: kb changed to R (gas constant)
If Cb = Ca then ln = 0
How do chemical reactions and free energy relate?
For a reaction to go forward - delta g for reactants must be higher than products (Forward < backward reaction)
What is the equilibrium constant?
Keq = [product] eq x [product]eq/ [reactant]eq x [reactant]eq
- fixed number
What is the mass action ratio?
r = [product][product]/[reactant][reactant]
- depends on actual conc
The difference between mass action ratio and Keq shows how far we are from equilibrium
What does delta G depend on?
Concentration independent terms - entropic
Concentration dependent terms
What are the features of delta g?
Free energy depends on concs
When at equilibrium = 0
Delta G values in tables are arbitrary - depend on conditions
Delta G will change for different sets of conditions
What is delta g ^o
Standard state - concs are all 1M - usually pH = 7 so we can ignore [h]
delta g = delta g^o + RTln([P]/[R])
If we let it get to equilibrium delta g = 0
Delta g^0 = - RTln([Products]eq/[reactants]eq)
= RTln(K)
How do we measure delta g^o
We let reaction reach equilibrium
0 = delta g ^o + RTlnk
delta g^o = -RTlnK
How do we calculate energy change for ions moving across a membrane?
= charge of object [coulombs] x PD (JC-1)
for H+ = delta G = e (electron charge) x membrane potential
for moles = x avogadro’s number
If we see volts as a change - x by charge on object = energy
What do reaction rates depend on?
Temperature
Pressure
pH
Ionic strength
Reagent conc
What is the irreversible unimolecular kinetic model?
A -> B
not reversible
Rate is proportional to concentration of A
v = -delta A/deltaT = deltaB/deltaT
rate = k[A] - FIRST ORDER
What is first order vs unimolecular?
First order - rates directly proportion to concentration
Unimolecular - A –> B
What are the units for k?
first order - usually min-1 or s-1
second - usually conc-1time-1
What is the reversible unimolecular kinetic model?
Rate of formation of B = k1[A] - k2[B]
What is the irreversible bimolecular kinetic model?
A + B –> C
second order
rate to form C = k[A][B]
A+B need to collide - and needs to be right orientation and violent enough
What is the average time a molecule stays in state a?
Average time = 1/k1
Same for B = 1/k2
What is Keq in relation to k1 and k2?
Keq = k1/k2
What is Keq when equilibrium shifts?
Keq = [product]/[reactant]
Keq gives you the proportion of how much of one state you have compared to another
e.g. we heat a reaction slightly, 100 um of folded is turned into 40 unfolded, 60 folded = 2/3
What is a way to work out the proportion of B?
[B]/[A]+[B]
this is the same as
([B]/[A])/ ([A]+[B])/[A]
this is the same as
Keq/1 + Keq
If we wanted A: 1/1+Keq
What is a bimolecular reversible reaction?
A + B -><- C
These include binding reactions: drug & receptor binding, TFs, enzyme/substrates
Rate of formation of PL = k1[P][L]
Rate of loss of PL = k-1[PL]
At equilibrium - rate of loss=formation
What is KD?
KD = koff/kon
can be [P][L]/[PL] or K-1/K1
Describes ligand binding
Stronger binding = lower KD
Units = 1/concentration
What is the fraction of bound protein?
[P][L]/[PL] = Kd
[PL]/[P]total = [L]/[L] + Kd
[P] total = [P] + [PL]
[L] total = [L] + [PL]
What are the rates of a bimolecular reversible reaction at equilibrium?
Rates equal
k1[P][L] = k-1[PL]
We write equilibrium as dissociation: PL -><- P + L
How does the fraction of [PL] change as [L] changes?
1) if [L] = 0.1Kd
[PL]/[P] total = 0.1/1.1 = 0.091 meaning 1 in 11 is PL
2) if [L] = 10 Kd
[PL]/[P] = 10/11 meaning 10 in 11 is PL
3) if [L] = Kd
[PL] /[P] = 1/2 meaning that there is 0.5 binding
What is the binding curve of [L] and fraction of [P] bound?
[PL]/[P]total on y axis
[L] on x axis
graph increases then plateaus - saturation point
What does a concentration-rate graph of a simple enzyme look like?
Rate increases but levels off at high substrate concentration as limited by enzyme conc
We assume we have a low conc of enzyme therefore [ES] must be very low so we can assume that [S]free = [S] total
What is the Michaelis Menten equation?
rate = kcat x [E]T x [S]/[S]+KM
kcat = catalytic constant - first order rate constant of ES –> E + P
KM = michaelis constant
Vmax = kcat x [E]T so we can simplify it to:
Vmax[S]/[S] + KM
If [s]» [km] then we can assume that [S] + Km = [S] - when we simply we get rate = Vmax
What is kcat?
first order rate constant describing the reaction of the ES complex to give products
ES –> E + P
What does kcat/KM mean?
Second order rate constant describing the reaction of E + S to give enzyme and products
What is KM?
The michaelis constant - the conc of substrate which produces half maximal rate - 1/2 vmax
Do simple enzymes have two steps?
Yes!
E + S –><– ES —> E + P
They reversibly make a complex ES which irreversibly makes a product
What is a competitive inhibitor and how does it affect Km and Kcat?
Bind to active site and prevent substrate binding
- we need a higher [S] so Km goes up
- once in active site it reacts normally so Kcat is the same
What is an allosteric inhibitor?
Binds to enzyme but not in active site - changes the active site shape
Can change either or both Km or Kcat - can increase or decrease
How can we measure enzyme activity?
We can measure the change of concentration by using a spectrophotometer
Beer-Lambert law: Abs = E x C x I
E = extinction coefficient (how strongly a molecule absorbs light)
C = concentration
I = path length of light through solution (usually 1cm)
What is arylsulphatase?
Accelerates release of sulphate from substrates
Nitrocatechol sulphate –> nitrocatechol
Yellow (at alkaline pH) –> bright red
We incubate the enzyme and substrate for 10 minutes then add NaOH to stop reaction and deprotonating nitrocatechol so it turns red
What happens when [S] is really small compared to Km?
Km + [S] = km
Vmax[S]/Km
Kcat/Km x [S] x [E]T - second order
What is the double reciprocal Menton graph?
Plotting 1/v (y axis) with 1/[s]
1/v = km + [S]/Vmax[S] = Km/Vmax[S] + 1/Vmax
1/v = Km/Vmax x 1/[S] + 1/Vmax
this is like y = mx + c
y intercept would be 1/Vmax
gradient = Km/Vmax
What makes a good signal?
Unique enough to relay a defined signal and only detected by correct receptors
Synthesised, released or altered quickly
Degraded quickly
What signalling do first messengers do?
Endocrine (hormones)
Paracrine (local mediators)
What are examples of endocrine signalling?
Adrenaline - aa derived - increases BP, HR and metabolism
Insulin - protein
Testosterone/oestrogen - steroid
What are examples of paracrine signalling?
Histamine - from mast cells - aa derived
ACh - from nerve terminals
How to cells receive many signals?
All receptors are proteins - 3D shape enable specificity
Affinity is mediated by non-covalent bonds
We want high affinity and high specificity
What is receptor activation and signalling?
Transmembrane protein - ligand binding causes conformative change - receptor is then activated and changes signal from extra to intracellular
What are the different classes of receptors?
Ligand gated
G-protein coupled
Enzymic
What is a ligand gated ion channel?
A multi-subunit pore where specific ions can pass - usually in synapses
What is the acetylcholine receptor?
A sodium channel - when bound subunits rotate to open pore
Causes contraction, learning/addiction (nicotine)
What are protein kinases?
The phosphorylation of AA side groups affects enzyme activity
Kinases can activate other kinases
What is receptor tyrosine kinase signalling?
RTK - ligand binding causes receptor dimerisation
Causes phosphorylation of cytosolic tyrosine AA’s - changes charge
Ligands are peptide e.g. insulin or growth hormone
Adapter proteins recognise and bind the phosphorylated tyrosines - activated intracellular pathways
Disorders can cause cancer
What is Ras GTPase switch?
When Ras (g protein) is activated (switches on) by RTK adapter proteins - Ras-GTP activates specific kinases needed for cell proliferation (division)
Ras GTPase hydrolyses GTP to GDP (turns Ras off)
Why is Ras important?
Important oncogene - some mutations lock Ras in the on state which can cause cancer
What causes calcium release?
RTK adapter proteins activate PLC - this cleaves a membrane phospholipid to produce inositol trisphosphate (IP3) and DAG from PIP2
This binds to ion channels on ER - releases calcium
What are G-protein-coupled receptors?
7 pass receptor
Ligand binding activated G protein - binds to GTP - moves away
Activate enzymes that produce secondary messengers e.g. PLC and adenylyl cyclase
Do GPCRs produce cAMP?
Yes!
Adenylyl cyclase produces cAMP
cAMP activates protein kinases - can boost energy release
What do receptors activate?
- cellular metabolism
- transcription
- cell division
- changes in cytoskeleton
What is the first law of thermodynamics?
Energy is conserved - it can neither be created nor destroyed
What does life require?
Negative entropy
What is catabolism and anabolism?
Anabolism = making stuff (endergonic - non-spontaneous - positive gibbs)
Catabolism = breaking stuff (exergonic - spontaneous - negative gibbs)
Catabolism provides the energy and precursors for anabolism
What is the difference between exergonic and endergonic?
Endergonic - requires the input of energy (anabolism)
Exergonic - releases energy (catabolism)
What are metabolic pathways?
Series of reactions - usually needing enzymes that catalyses the conversion of one molecule to another
What must a metabolic pathway be?
- physically possible
- thermodynamically likely (- gibbs)
- kinetically feasible
- shielded from unwanted side reactions
What are the benefits of metabolic pathways?
- make complex transformations kinetically possible
- allow multiple energy producing sites by releasing energy in packets = can be coupled to carrier molecule
- generate chemical structures
- allow high level of control (more steps = more control)
What are common features of metabolism?
- many common pathways
- 6 basic types of reaction
- common organisation patterns
- common regulatory principles
- common co-factors
- use ATP
What are the 6 basic types of reaction?
1) Oxidation-reduction - e transfer by oxidoreductases
2) Ligation (using ATP) - forming bonds by ligases
3) Isomerization - rearranging atoms to form isomers by isomerases
4) Group transfer by transferases
5) Hydrolytic - adding water to cleave bond - hydrolases
6) Adding or removing groups - lyases
What are the common organisational paradigms (patterns)?
1) physically separate soluble enzymes with diffusing intermediates
2) a multienzyme complex - metabolons - substrates channel between them before product release
3) membrane bound multienzyme system
These can be compartmentalised into organelles or in proteinaceous compartments (prokaryotes)
What are the benefits of compartmentalisation?
- substrate channelling (substrate moved directly from one active site to another)
- increases rate as the conc is increased
- avoids unwanted side reactions and futile cycling
How are metabolic pathways studied?
- cell fractionation
- inhibitors
- radiolabelling
- mutants
What is cell fractionation?
We break cells with high freq sound and force cells through small hole using high pressure
Then do density ultracentrifugation - separates cellular components - test which have particular enzyme of interest
How can we use inhibitors to study metabolic pathways?
We can use an inhibitor and what we identify shows what has inhibitive properties
- if reactants build up - a step has been inhibited
- can bind to proteins so enzymes can be identified
What did radiotracing allow us to do?
Allowed us to discover the calvin cycle
Algae was supplied with 14CO2 - then illuminated. The reaction is stopped by draining the contents into hot alcohol.
Then do 2 solvent chromatography - those with radioactivity were identified.
A 10 seconds - most radioactivity was in 3-phosphoglycerate, at 2 minutes phosphorylated glucose and fructose were identified
How can we study metabolism using mutants?
We can knock out specific genes to prevent a certain protein being produced.
We can see what steps are not being catalysed e.g. B is building up.
We then could add C which would restore the ability to make F
How do we obtain energy from reduced energy sources?
We could either use a stepwise oxidative approach where we overcome many small activation energies using the bodies heat - energy stored in a carrier molecule
Or we could overcome a large activation energy from heat from a fire - we would release energy as heat
We use stepwise as it is controlled and allows us to capture energy in carrier molecules
What atom is reduced in carbon dioxide?
Oxygen, carbon is oxidised
This is because the electrons are unevenly shared
In methane - carbon is reduced
What bonds between C-H, C-O and C–O is least stable?
C-H so energy is released when replaced by C-O or C–O
What happens to the free energy released during oxidation?
Coupled to the generation of activated carrier molecules - can drive endergonic reactions
What are some common activated carrier molecules?
ATP - phosphate
NADPH/NADH - hydrogen and e
Carboxylated biotin - carboxyl group
S-adenosylmethionine - methyl group
Uridine diphosphate glucose - glucose
What is more stable: ATP or ADP and pi?
ADP - ATP is less stable
This is because the phosphates are negatively charged and repel
The entropy is increased in ADP and Pi
Water stabilises ADP and Pi more
The free Pi is stabilised by resonance
This means that equilibrium favours ADP + Pi, k (equilibrium constant - ratio of products to reactants) = 1,000,000 - a million more ADP and Pi
What is the free energy change of ATP?
Delta g = RT ln(mass action ratio/k)
K = equilibrium constant
In a dead cell MAR = k - delta g is 0 so k = 1,000,000
When MAR<k - delta g is negative
When MAR>k - delta g is positive
Are co-factors kinetically stable?
Yes!
All react slowly with water/oxygen - there is a large activation energy barrier in absence of enzymatic catalyst
They are stable as it allows enzymes to control flow of energy and reducing power
Is free energy coupled in metabolism?
Say free energy is negative for y –> x - this reaction can happen spontaneously
x –> y is not favourable so is coupled to a second energetically favourable reaction
Anabolic (some catabolic) involve coupling endergonic reactions to exogonic so net delta g is negative
What is glycolysis?
First step of sugar oxidation
No O2 required but oxidation is involved to make 2NADH
Some ATP required - net makes 2 ATP
Known as EMP pathway
NEED TO KNOW STRUCTURE?
What is glycolysis step one?
Glucose phosphorylation - the Pi on glucose traps the G6P in the cell - also keeps the conc of glucose low in the cell - promotes glucose transporter uptake
Hexokinase does this - binds to glucose which causes a conformative change - active site around glucose and ATP becomes more non-polar - gets rid of water
This favours transfer of Pi from ATP to glucose and prevents hydrolysis of ATP by water
This is an example of substrate induced fit and enzymatic coupling
How does hexokinase phosphorylate glucose?
The active site has an aspartate which deprotonates the C6 hydroxyl group
the -O: acts as a nucleophile attacts gamma Pi of ATP
Does not need water
Lowers activation energy
What is the free energy of a coupled reaction?
Two reactions can be coupled if they share one or two intermediates e.g. Glucose + Pi -> glucose phosphate (+ delta g) and ATP -> ADP + Pi (negative delta g)
The free energy change is the sum of individual reactions
How does coupling effect the equilibrium constant?
The delta G has changed
K = e^ - (delta g/RT)
What is the second step of glycolysis?
Glucose-6-phosphate is isomerased by phosphoglucose isomerase to fructose-6-phosphate
This is reversible
This forms a ketose sugar (fructose) from an aldose sugar
What is the third step of glycolysis?
Fructose-6-phosphate is phosphorylated to fructose-1,6,-bisphosphate by phosphofructokinase using ATP
This Pi destabilises the sugar promoting cleavage in step 4
Entry of sugars into glycolysis is controlled by allosteric regulation of phosphofructokinase
What is flux in ATP?
Cells use energy from food to constantly make ATP - this maintains the mass ratio ([ADP] [Pi]/[ATP]) a long way from equilibrium allowing it to act as an energy store
What regulates glycolysis?
G6P will reduce hexokinase by negative feedback
ATP allosterically dials down phosphofructokinase and pyruvate kinase
When there’s too much ATP - slow down glycolysis
Whilst exercising, AMP will allosterically promotes phosphofructokinase - more ATP made
What is step 4 of glycolysis?
Fructose-1,6-bisphosphate is cleaves by aldolase to form dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate (GAP)
Only GAP can go through glycolysis
What is step 5 of glycolysis?
DHAP is converted to GAP by triose phosphate isomerase (TIM)
TIM is a kinetically perfect enzyme limited by how fast the substrate moves in and out of active site
What is TIM?
Triose phosphate isomerase
Suppresses formation of toxic intermediate methyl glyoxal from enediol intermediate
Achieves this by movement of 10 AA loop region over active site blocking exit until GAP is formed
H from the first carbon on DHAP goes to Glu 165 - enediol intermediate
then another H from the first carbon OH goes to His95, then the O- on DHAP turns into a double bond then the H from Glu165 goes to the middle carbon - GAP
What are the products of glycolysis?
2 x pyruvate
2 x ATP
2 x NADH
What is step 6 of glycolysis?
Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) uses the coenzyme NAD+ to oxidise GAP forming NADH
First step - oxidation of an aldehyde to acid (favourable) - second forms acyl-phosphate group (unfavourable) - so this is coupled by formation of enzyme-bound thioester intermediate - if wasn’t coupled it wouldn’t happen as the first drives the second - first steps energy is used for second
The formation of the thioester intermediate reduces the activation energy to form 1,3-BIS
GAPDH couples oxidation to transfer Pi to the sugar forming 1,3-bisphosphoglycerate
First energy generating step where NADH is formed
We don’t want to form carboxyl group as it is energetically unfavourable to add phosphate - so we make the intermediate
What is NADH?
Nicotinamide adenine dinucleotide
Energy released from carbon oxidation forms NADH - needs 2 e and a proton
NADH is used by ETC to make ATP by oxidative phosphorylation
What is step 7 of glycolysis?
Phosphoglycerate transfers a Pi to ADP from 1,3 BPG - substrate level phosphorylation
Makes 3-phosphoglycerate
Free energy change is -18.9 but runs near equilibrium as [ATP] is high in cytoplasm
Mg in active site activates ADP for reaction
What is step 8 of glycolysis?
Phosphoglycerate mutase transfers phosphoester linkage from carbon 3 to 2
Forms 2-phosphoglycerate
What is step 9 of glycolysis?
Enolase removes water from 2-phosphoglycerate - forms phosphoenolpyruvate
2PG and PEP have same potential energy so enolase rearranges substrate into a form where more energy can be released upon phosphoryl transfer
What is step 10 in glycolysis?
Pyruvate kinase transfers a phosphate to ADP and forms pyruvate from phosphoenolpyruvate
This reaction is far from equilibrium so pyruvate conc is kept low
What happens after glycolysis?
O2: converted to acetyl-coA via link reaction
No O2: pyruvate fermented to lactate
What is fermentation?
No O2 - NADH builds up so used to reduce pyruvate to lactate or ethanol - this regenerates NAD+ to restore REDOX balance and allow glycolysis to continue - reduces middle carbon from 2+ to 0
Lactose dehydrogenase does this in animals
In yeast - 2CO2 made (pyruvate decarboxylase) to form 2 acetaldehyde then NADH donates hydrogen to form 2 ethanol (alcohol dehydrogenase)
What enzymes drive the big energy changes in glycolysis?
Hexokinase
Phosphofructokinase
Pyruvate kinase
What is a redox potential?
A measure of the ability of a redox couple to accept electrons
Couples with very negative redox potential = good e donors (reductants)
Very positive - good e acceptors (oxidants)
How are electrons transported in the ETC?
NADH - reduces complex I
Succinate/fumarate - reduces complex II
Complex I and II transfer these to UQ which carry e to complex III - reduce cytochrome C - then pass to complex IV to reduce H2O/O2
The free energy from the ETC pumps hydrogen in
We can use DCPIP in place of O2 to measure activity - turns from blue to colourless - between complex III and IV
What are some inhibitors of the ETC?
Complex I - barbitone
Complex III - antimycin
Complex II - malonate
Complex IV - azide
What happens to pyruvate after glycolysis in presence on O2?
Will be converted to Acetyl Co-enzyme A via link reaction
Acetyl CoA is metabolised in the kreb cycle
These reactions form CO2
What is the link reaction?
Pyruvate –> Acetyl CoA + CO2
This requires pyruvate dehydrogenase complex which contains 3 enzymes: pyruvate decarboxylase, dihydrolipoyl transacetylase and dihydrolipoyl dehydrogenase
An NADH is also made
What is pyruvate dehydrogenase?
Enzyme complex in link reaction
Binds 5 cofactors:
- thiamine pyrophosphate
- lipoamide
- FAD/FADH2
- CoA
- NAD/NADH
What is CoASH?
An activated carrier molecule carrying an acetyl group by thioester linkage
Hydrolysis of thioester linkage is energetically favourable (- delta g)
This can be coupled to reactions with positive delta g - e.g. acetyl transfer to oxaloacetate
What is FAD/FADH2?
Flavin adenine di-nucleotide
Common activated carrier molecule capable of carrying 2 electrons and 2 protons
What is pyruvate dehydrogenase?
In step one of link reaction - pyruvate is decarboxylated - makes an hydroxy-ethyl fragment (BY PYRUVATE DECARBOXYLATE (E1))
This fragment is bound to TPP cofactor
In step 2, hydroxy-ethyl-TPP is oxidised to acetyl fragment by lipoamide cofactor on E2 (DIHYDROLIPOYL TRANSACETYLASE) - this forms acetyl-dihydrolipoamide
In step 3, acetyl-dihydrolipoamide reacts with coASH to form acetyl-CoA and dihydrolipoamide. FAD and NAD is oxidised to NADH and FADH2
What is the first step of the kreb cycle?
Citrate synthase removes a proton from the methyl group on acetyl-CoA
This forms a CH2- which acts as a nucleophile towards carbonyl group on oxaloacetate
Hydrolysis of CoA-intermediate drives forward reaction as energetically favourable + water - HS-CoA and H + is removed
Forms citrate
What is step 2 of kreb cycle?
Aconitase isomerases citrate (tertiary alcohol) to isocitrate (secondary alcohol)
This is by removing water (OH from middle carbon) then adding it back to end carbon
Makes next step easier as we break C-H instead of C-C
What is step 3 of kreb cycle?
This is the first oxidation step
Isocitrate dehydrogenase catalyses oxidation of the 4th carbon - hydroxyl to carbonyl
An NADH is formed
The intermediate formed is unstable so is decarboxylated to alpha-ketoglutarate and CO2
Why is CO2 important?
Decarboxylation gives a strong thermodynamic pull to a reaction as:
- very stable
- easily escapes (soluble)
- more products than reactants so positive entropy
What is step 4 of the Kreb cycle?
Alpha ketoglutarate dehydrogenase catalysed oxidation of carbon 5 from +3 to +4 by - decarboxylation - carbon 4 is oxidised from +2 to +3
Add HS-CoA
This is coupled to formation of NADH and Succinyl CoA
CO2 is formed
What is step 5 of kreb cycle?
Succinyl-CoA synthetase catalyses the hydrolysis of the thioester bond and replacement with phosphodiester bond forming succinyl phosphate (-ve delta g)
The phosphate is then transferred to ADP to form ATP - substrate level phosphorylation
Forms succinate
How does succinyl-CoA synthetase work?
CoA is displaced by phosphate - succinyl phosphate.
Histidine then removes Pi forming succinate and phosphohistidine - phosphate then transferred to ADP - ATP (substrate level phosphorylation)
What is step 6 of kreb cycle?
Succinate dehydrogenase (transmembrane protein in inner mito mem) uses FAD to oxidise succinate to fumarate - FADH2
FAD is reduced as the free energy is insufficient to reduce NAD+
What is step 7 of kreb cycle?
Fumarase converts fumarate to malate by adding water across c=c bond
What is step 8 of kreb cycle?
Malate dehydrogenase uses NAD+ to converts OH on malate to a carbonyl group
Makes oxaloacetate
What is an amphibolic cycle?
Catabolic and anabolic
e.g. kreb cycle
How was kreb cycle uncovered?
He observed that adding malate/citrate/succinate or fumarate to minced pigeons - stim a lot of oxygen
If intermediates are low in supply - rate of pyruvate oxidation is limited - adding any intermediate caused high O2 release as more NADH and FADH2
He saw that adding malonate competively inhibited respiration - inhibiting succinate dehydrogenase so build up of succinate
Adding any poison caused succinate accumulation - must be a cycle
What is the warburg manometer?
Measures changed in pressure caused by oxygen uptake
CO2 is absorbed by filter paper soaked in KOH
Substrate added by side flask
How can we make acetyl CoA from fats?
Fatty acid beta oxidation - form more energy than sugars as they are more reduced
Lipases break down fats into glycerol and FAs - glycerol enters glycolysis (converted to DHAP), FAs activate by linkage to CoA and are transported into mitochondria
The fatty acids CoA is oxidised by 4 enzymes (- delta g) and form NADH/FADH2 (+ delta g) (can be used in oxid phos) and acetyl CoA
What are the electron donors in the ETC?
NADH (complex I) and FADH2 (complex II - succinate dehydrogenase) pass electrons to ubiquinol to make ubiquinone which transfers to complex III - this then transfers to cytochrome c to then complex IV - the electrons then transfer to oxygen
Free energy is used to drive formation of proton motive force for ATP synthesis
How can we study the mitochondria?
Isolate the mitochondria by cell disruption and centrifugation
Plunge freeze into liquid ethanol on a grid
Cryo-electron microscopy searches the grid then images are taken at small tilt increments
The images are aligned and reconstruct the microscopy
How do electrons flow in ETC?
Through redox reactions - redox potentials
What are redox potentials?
Measure of the affinity of a redox couple for electrons
More negative = more likely to donate
More positive = more likely to accept
What is chemiosmotic coupling?
Free energy released as electrons travel from negative to positive redox potentials - used to move protons from matrix to IMS (low to high)
What is the free energy in the redox reaction in the ETC?
Downhill (-ve delta g) - energy used to couple to proton transport
What is a standard redox potential?
Measured using the compound with a standard hydrogen half cell containing 10-7 M h+ and 1atm of hydrogen gas - with a salt bridge
If electrons move from hydrogen to compound - has positive redox potential
If electrons move from compound to hydrogen - negative redox potential
What are some redox potentials in the ETC?
NADH -> NAD + H + e = -320 mV
FADH2 -> FAD + 2H + 2e = -30
UQH2 -> UQ + 2H +2E = +50
CytCred -> CytCox + e = 250
H2O -> O2 + 2H + 2e = 870
How do we work out delta g from redox potentials?
We work out change in redox potential - standard redox pot acceptor - donor
Delta G = z x F x redox potential change
F = faraday constant - 96485 Jmol
z = number of charges transfered
Redox potential- make sure in volts
How do we calculate the actual redox potential?
Em = Em0 + (RT/nF) ln([ox]/[red])
n = number of e transfered
CHANGE mV to V
What is complex I?
NADH dehydrogenase
Oxidises NADH to NAD+ - transfers to ubiquinol - used to pump 4H+
What is succinate dehydrogenase?
Complex II
It oxidises succinate to fumarate in the kreb cycle - electrons go to FAD
These two electrons then reduce UQ to UQH2
NO protons are pumped
What is ubiquinone?
Coenzyme Q10
A lipid soluble electron carrier - takes e from complex I and II to III
It takes up H+ from matrix when reduced and releases them into the IMS when oxidised
Which complexes pump H+ into the IMS?
Complex I and IV
NADH and UQ bind to complex I - conformational change that promotes H+ uptake into the complex
UQ reduction causes another change that changes the side where H+ is bound to
Release of NAD and UQ2 causes drop of affinity - releases into IMS
What else feed UQ?
FADH2 from fatty acid B oxidation (fatty acid CoA dehydrogenase) and NADH from glycolysis (glycerol 3-phosphate dehydrogenase)
What is cytochrome bc1?
Complex III - oxidises UQH2 to UQ by transferring electrons to cytochrome c, The free energy translocates 4H+ into IMS
UQH2 + 2cyt cox + 2H+ (matrix) –> UQ + 2cyt cred + 4H+ (ims)
How do complex I & II interact with III?
Complex III oxidises UQH2 to UQ
UQH2 is provided by complex I or II
2H+ is taken from matrix when complex I/II convert UQ and released when complex III oxidises it
Another 2H+ are pumped in
What is the structure of cytochrome c?
A small soluble electron carrier
In intermembrane space
Each cytochrome c binds to one electron which reduces Fe3+ to Fe2+
What is complex IV?
Cytochrome C oxidase
Transfers electrons from cyt c to O2 - needs 2 electrons and 2 hydrogen ions - makes h+
The free energy pumps 2 H+ in
What is complex V?
ATP synthase
The energy in the proton motive force drives the energetically unfavourable reaction of making ATP
One full turn of Fo motor carries 12H+ causing a full turn of F1 ATPase forming 3 ATP
How do we calculate the free energy of the PMF?
Pmf is a combination of membrane potential and proton conc gradient
pmf = membrane potential (trident) - 2.3(RT/F x (change in pH))
Delta g = zF(pmf)
What is the efficiency of forming ATP by pmf?
Delta g of making one ATP = 46kJ mol
4H+ needed for one ATP
3 x ATP = 138 kJmol
12 x -17.5 = 210 kJmol
138/210 = 66%
How many H+ are transferred per cofactor?
For each NADH = 10H+
For each FADH2 = 6H+
2.5 ATP per NADH
1.5 ATP per FADH2
How many ATP do we make per glucose?
30
What is the chemiosmotic hypothesis?
Peter Mitchell suggested that the electrochemical proton gradient generated by electron transport was used to generate ATP
What is evidence for chemiosmosis?
- when mitochondria respire, the ratio of H+ between matrix and IMS changed - electron transport was coupled to change in osmotic potential
- the proton gradient was abolished by an uncoupler e.g. DNP (allowed proton diffusion)
- when adding a proton pump in mitochondria instead of ATP synthase caused ATP - showed there was no high energy intermediates
How does Γ and k effect delta g?
Γ = product conc
k = equilibrium product conc
Γ«_space;K and Γ/K <1 then delta g is more negative
Γ» K and Γ/K >1 then delta g is more positive
What does a low pka mean?
Strong acid - wants to give away proton
What are macrostates and microstates?
Macro - a collection of microstates representing a system as a whole
Micro - specific configuration within a system - different possible arrangements of position and energy
Most likely macrostate will be a balance between the likelihood of microstates and how many there is
