Biochemistry 1 Flashcards

1
Q

What are the electron orbitals?

A

s = 2
p = 6
d = 10
f = 14

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2
Q

How many electrons in each shell?

A

1st = 2
2nd = 8
3rd = 18
4th = 32

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3
Q

What is the shape of the 2p orbitals?

A

2pz = /
2py = l
2px = –
x then y then z

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4
Q

What is the nodal plane?

A

The region around a nucleus which the probability of finding an electron is zero.

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5
Q

What is the electron octet?

A

Noble gas configuration.
Full outer shell.
E.g. F gains an electron from Na - both will have electron octet

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6
Q

What are waves?

A

Electrons can be presented as waves - in phase = same wave.

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7
Q

What is a covalent bond?

A

2 electrons shared in 2 overlapping orbitals from 2 atoms with orbitals of similar energy.

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8
Q

What happens when 2 atomic orbitals combine in phase?

A

They form a bonding orbital which is lower than the original orbitals = bonding molecular orbital.

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9
Q

What happens when 2 atomic orbitals combine out of phase?

A

They form an antibonding molecular orbital which is higher in energy.

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10
Q

What is a sigma bond?

A

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.

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11
Q

What is a pi bond?

A

Formed when two p orbitals overlap laterally - NOT ON BOND AXIS.
Usually occurs in addition to sigma - in double/triple.
Restrict rotation and weaker.

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12
Q

What is a hybrid orbital?

A

An orbital formed by combination of two or more atomic orbitals.

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13
Q

What is sp3?

A

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.

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14
Q

What are the bonds in methane?

A

Sigma bond - 1s orbital from H overlaps with sp3 of C

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15
Q

What are examples of sp3 hybridized atoms?

A

Oxygen in water
Nitrogen in ammonia
1s of H overlaps the sp3 orbital, some sp3 orbitals have lone pairs

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16
Q

What is a molecular orbital?

A

Combination of atomic orbitals of similar energy - can be bonding or antibonding

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17
Q

What does in and out of phase mean?

A

In = electron waves are the same
Out = electron waves are different

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18
Q

What is an ionic bond?

A

Electron transferred - orbitals far apart in energy.

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19
Q

What does the excited state mean?

A

Ground - normal
Excited - moves to higher energy level e.g. s electrons move to p = hybridisation to sp3

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20
Q

Why can oxygen bind to 2 things?

A

The electrons are excited to sp3 orbitals - two have 2 electrons, 2 have 1 electron so can only make 2 covalent bonds.

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21
Q

How is the c-c bond formed in ethane?

A

Overlap of 2 carbon sp3 orbitals = sigma

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22
Q

What is an sp2 orbital?

A

Trigonal arrangement
1 s blends with 2 p

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23
Q

What is the bonding in ethene?

A

Sigma bond between c-c (sp2-sp2)
Sigma bond between c-h (sp2-s)
pi bond between c-c (p-p)

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24
Q

What is the bonding ethyne?

A

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

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25
Q

What are the bond angles?

A

single c-c = 109.6
double c-c = 121.7
triple c-c = 180

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26
Q

What is non-polar covalent bonding?

A

Equal sharing of electrons

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27
Q

What is polar covalent bonding?

A

Sharing electrons between atoms of different electronegativities.

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28
Q

What is the difference between sp2 vs sp3?

A

sp2 = one s and two p = 3 sp2
sp3 = one s and three p = 4 sp3

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29
Q

What does the A with a circle on top mean?

A

0.1 nm
Angstrum

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30
Q

What are the shapes of sp orbitals?

A

sp = linear
sp2 = trigonal
sp3 = tetrahedral

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31
Q

What effect does s character have?

A

More character - shorter, stronger and larger bond angle

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32
Q

What aa’s are proteins made of?

A

L amino acids (d is an isomer but not used - but just as good)

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33
Q

What types of side chains do amino acids have?

A

Non polar
Polar
Polar positively charged
Polar negatively charged

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34
Q

What is a disulfide bond?

A

Between two cysteines to make a cystine

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35
Q

What direction do polypeptides go?

A

Written from N to C terminus

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36
Q

What are the features of a peptide bond?

A

Very stable and planar (can’t move due to partial double bond character)
Partial double character
Cleaved by proteolytic enzymes
c–o = sp2

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37
Q

What rotation is in amino acids?

A

N-C = phi (line in circle)
C-C = psi (trident)

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38
Q

What did Ramachandran say?

A

Many combinations of phi and psi are not found because of steric clashes

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39
Q

What are the bond lengths?

A

Single = 1.54 A
Double = 1.33 A
Triple = 1.20 A

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40
Q

Why can’t the peptide bond rotate?

A

Due to the delocalisation of electrons from the double-bonded oxygen to the peptide bond.

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41
Q

What is the denaturation of ribonuclease A?

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.

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42
Q

What is the free energy required to denature AA’s?

A

0.4 kJ/mol per Amino Acid

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43
Q

What free energy is needed to overcome hydrogen bonds?

A

12 kJ/mol

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44
Q

What group does cysteine have?

A

SH = thiol

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45
Q

What covalent bond do adjacent cysteines make?

A

A disulphide bond s-s
Requires oxidative conditions
Only bond formed between side chains
Can provide extra stability

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46
Q

What are the non-covalent forces which hold proteins together?

A
  • ionic interaction
  • van der waal interactions
  • hydrogen bond
  • hydrophobic effect
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47
Q

What is the energy of association?

A

E = k q1 q2 /Dr
q1 & 2 = electric charges
r = distance
k = 9 x 10^9 JmC-2
D = dielectric constant

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48
Q

What is one electrical charge?

A

1.6 x 10^-19 C

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49
Q

What is D?

A

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

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50
Q

What are the three wan der Waal interactions?

A

Dipole-dipole interactions
Dipole-induced dipole interactions
London dispersion forces

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51
Q

What is a dipole-dipole interaction?

A

Occur between polar molecules which have permanent dipoles.
e.g. between c–o, c–o

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52
Q

What is a dipole-induced dipole interaction?

A

Between polar and non-polar (creates temporary dipole)
e.g. c–o, h3c

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53
Q

What is a london dispersion force?

A

Present in all molecules due to fluctuating asymmetric distribution of electrons.
e.g. ch3, ch3

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54
Q

What is a hydrogen bond?

A

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

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55
Q

What is the energy association of non-covalent interactions?

A

Hydrogen bonding = 4-13 kJ/mol
Ionic interactions = 5 kJ/mol
Van der waal = 2 kJ/mol

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56
Q

Which is longer: covalent or hydrogen bonding?

A

Hydrogen

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57
Q

What is the hydrophobic effect?

A

Influences which cause nonpolar substances to minimise their interaction with water - form micelles in aqueous solutions.
Non-polar

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58
Q

What can water solubilise?

A

Polar, ionic and hydrophilic

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59
Q

How does water act with hydrophobic parts?

A

Forms ‘cages’.
When buried - caged water molecules are released which increases entropy.

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60
Q

How do proteins fold?

A

Spontaneous - gibbs needs to be negative.
Enthalpy change is slightly negative.
Entropy change is positive as caged water is released.

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61
Q

What is the gibbs equation?

A

^g = ^h - t^s
gibbs free energy = enthalpy change - T entropy change
negative = feasible

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62
Q

Which non-covalent interactions stabilise proteins?

A

Hydrogen bonds = strong
Ionic = strong but don’t stabilise well
Dipole-dipole = weak but stabilise well

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63
Q

Why do proteins need to fold?

A

Residues need to come close to eachother to help function

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64
Q

NEED TO KNOW ALL PROTEINS?

A
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65
Q

What is the primary structure of a protein?

A

Linear sequence on amino acids
From N terminus to C terminus

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66
Q

What is the secondary structure of a protein?

A

Stabilised by hydrogen bonding
- alpha helix and beta pleated sheet

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67
Q

What is an alpha helix?

A

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

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68
Q

What is the alpha helix terminator?

A

Pro (proline)

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69
Q

Are alpha helices hydrophobic or hydrophilic?

A

Amphiphilic - have both
Helix has a hydrophobic and hydrophilic sides
Occur in globular proteins - hydrophobic face interior, hydrophilic face solvent.

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70
Q

What is a beta sheet?

A

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.

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71
Q

What is the supersecondary structure of a protein?

A

Combination of secondary structures (e.g. beta-alpha-beta, alpha-alpha)

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72
Q

What is the tertiary structure?

A

Assembly of secondary elements into native protein structure

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73
Q

What is the quaternary structure of a protein?

A

Multiple polypeptide chains assemblied
homooligomer - identical monomers
heterooligomer - different

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74
Q

What is an allosteric interaction?

A

Ligand binds to quaternary structure - alter affinity of ligand to another subunit

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75
Q

What are domains?

A

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)

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76
Q

What is an immunoglobulin?

A

Each domain in IgG is similar - antiparallel beta sheets surrounding hydrophobic core

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77
Q

What is a conformative change?

A

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.

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78
Q

What is phophorylation?

A

OH of ser, thr and Tyr can be reversibly phosphorylated

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79
Q

What is glycosylation?

A

Addition of carbohydrates - increases hydrophilicity and ability to interact with other molecules.

On asn or can be ser and thr

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80
Q

What is hydroxyproline?

A

OH group added to proline - this stabilises collagen fibres - Vit C deficiency can inhibit this

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81
Q

What is y-carboxyglutamate?

A

Vitamin K deficiency can result in low carboxylation of glutamate in prothrombin - can cause haemorrhage.

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82
Q

What are protein families?

A

AAs with a closely related amino acid sequence - arises from common ancestor

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83
Q

What are serine proteases?

A

Family of enzymes which all contain - asp-his-ser
Includes: chymotrypsin, trypsin and elastase
Have very similar structures and include digestive enzymes and blood clotting

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84
Q

What is a reaction mechanism?

A

1) determine where the electrons are
2) determine what bonds are made/broken
3) describe flow of electrons

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85
Q

What is a lewis structure?

A

1) draw molecular skeleton
2) assume bonds are covalent
3) count electrons
4) add sigma bonds
5) add pi if necessary

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86
Q

What is an electro and nucleophile?

A

Electrophile - accepts electrons
Nucleophile - supplies electrons

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87
Q

How would we draw a sigma bond being made?

A

e.g. when an anion and cation encounter
Arrow is from lone pair of electrons to where they move to.
Reversible

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88
Q

What is substitution nucleophilic bimolecular?

A

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

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89
Q

What is a lysozyme?

A

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

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90
Q

How do lysozymes work?

A

Cuts a glycosidic bond between NAM and NAG sugars (have similar structure) in peptidoglycan
Glycosidic bond between 4th and 5th sugars break

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91
Q

What is the structure of a lysozyme?

A

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

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92
Q

What is the lysosyme active site?

A

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)

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93
Q

What is the lysozyme mechanism?

A

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

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94
Q

What are the products of a lysozyme reaction

A

Sugars E,F
Sugars ABCD

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95
Q

What do enzymes do?

A

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

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96
Q

What is the difference between pH, Ka and pKa?

A

pH = conc of hydrogen ions
pKa = strength of acid
ka = dissociation constant

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97
Q

What is the dissociation constant?

A

Ka = [H][A]/[HA]

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98
Q

What is pKa?

A

pKa = - log(Ka)
pKa basically turns Ka into a nice number

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99
Q

What is the Henderson-Hasselbalch equation?

A

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

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100
Q

How does pH change as hydroxide is added?

A

pH increases - cation (nh3+) then zwitterion then anion (coo-)
Some plateaus in graph - pK1, pl (around 6-9), pK2

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101
Q

What is pl?

A

Isoelectric point - zwitterion

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102
Q

How many pKa values in an amino acid?

A

2 or 3 if the side chain is titratable
Amino group - pKa = 9-10
Carboxyl group - pKa = 2-3

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103
Q

What effect does the side chain of histadine have on pKa?

A

The chain has a pKa of 6 - makes it very sensitive to pH under normal conditions

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104
Q

Are most enzyme reactions pH sensitive?

A

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

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105
Q

Why can the local environment influence sidechain pKa?

A

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

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106
Q

What is the name of a protonated and unprotonated form of the histidine side chain?

A

Protonated = imidazolium ion
Unprotonated = Imidazole

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107
Q

What groups do papain have?

A

Cys25 - wants to be unprotonated - 4.2
His15 - wants to be protonated - 8.2
Has bell-shaped curve

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108
Q

How does an enzyme make a reaction faster?

A

Turns a intermolecular reaction into a faster intramolecular one.
Holds them in an optimum orientation so they have react.
This is rate enhancement

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109
Q

Are there intermediates from substrate to product?

A

Yes!
There may be intermediates (lower curve) or transition states (high curves) - intermediates are more stable.
Enzymes prefer to bind transition states

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110
Q

How do enzymes bind to substrates?

A

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’

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111
Q

How does the induced fit theory work?

A

Substrate binds non-optimally and is stressed when bound - enzyme strained but the strain energy is released when transition state is reached

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112
Q

What is catalysis?

A

The process that increases the rate at which a reaction approaches equilibrium

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113
Q

What steps occur in enzyme catalyzed reactions?

A

1) general acid-base catalysis - donation/gain of proton
2) covalent catalysis
3) metal ions in catalysis

Lower free energy pathway

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114
Q

What is an example of acid-base catalysis?

A

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

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115
Q

What is Vmax and km?

A

Vmax = maximum velocity (rate)
Km = 1/2 Vmax

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116
Q

What are enzyme inhibitors usually?

A

Transition state analogs

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117
Q

What is covalent catalysis?

A

Some enzymes can form covalent bonds with substrates - generate impermanent intermediates

Usually involves a strong nucleophile on enzyme

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118
Q

How does a lysozyme use covalent catalysis?

A

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

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119
Q

What metal ions are used in catalysis?

A

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

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120
Q

How are metals used in catalysis?

A
  • 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+
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121
Q

What are cofactors?

A

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)

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122
Q

What are coenzymes?

A

Small organic cofactors

can be tightly bound (prosthetic group)
can be loose or can dissociate (cosubstrate)

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123
Q

What are the cofactor + enzyme called?

A

Enzyme alone = apoenzyme
Together = holoenzyme

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124
Q

Can proteins help enzymes?

A

Yes!
They are a protein coenzyme and usually involved in transport

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125
Q

What is AMP?

A

Adenosine monophosphate
- building block in cofactors
- phophate group, adenine ring (2) and ribose ring

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126
Q

What are nucleoside triphosphates?

A

ATP, GTP (guanine) ect.
- know structure

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127
Q

What is NAD?

A

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)!!!

128
Q

What are the different classes of enzymes?

A

Oxidoreductases - redox
Transferases - transfer things
Hydrolases - transfer things involving water
Lyases - make double bond
Isomerases - intramolecular group transfer
Ligases - joining molecules by ATP

129
Q

What are oxidoreductases?

A

Often use a cofactor e.g. NAD
Carries out REDOX
Half equations can show this

130
Q

What are transferases?

A

Transfers a group between molecules e.g. nucleophilic substitution

Group transfer reactions - transfer of an electrophile from one nucleophile to another

131
Q

What is a group transfer reaction?

A

Transfer of electrophiles from one nucleophile to another

132
Q

What is a hydrolase enzyme?

A

Cleavage reaction via addition of water
e.g. aryl sulphatase

133
Q

What is a lyase enzyme?

A

Also known as synthases
Add or remove groups to make double bond
e.g. enolase

134
Q

What is an isomerase enzyme?

A

Interconversion of isomeric forms of compounds
When converting L-AA to D-AA - racemization

135
Q

What is a ligase enzyme?

A

Joining two molecules requiring ATP
e.g. CO2 + pyruvate –> oxaloacetate needs pyruvate carboxylase - requires biotin cofactor

136
Q

What is a membranes permeability?

A

Permeable: gases, ethanol
Slightly permeable: water, urea (small uncharged polar)
Permeable: glucose, ions, charged polar molecules (AA, ATP)

137
Q

What fats are in membranes?

A

Phospholipids - glycerol + 2 FAs + phosphate attached to alcohol

  • phosphatidyl serine
  • phosphotidyl choline
  • phosphotidyl ethanolamine
  • phosphotidyl inositol

Know structure??

Can influence fluidity, curvature and thickness

138
Q

How does membrane composition change between cell types?

A

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

139
Q

What does cholesterol do to membranes?

A

Makes them thicker as it has a lipid-ordering effect
Has hydrophilic (OH at end) and phobic parts (steroid rings)

140
Q

What causes membrane curvature?

A

Segregation of lipids - depends on size of heads and tails
P-choline - cylindrical lipids - bilayers flat
P-ethanolamine - cone shaped - curved

141
Q

What determines membrane fluidity?

A

Fatty acid composition and cholesterol
Cholesterol - stiffens the structure and makes thicker

142
Q

How much proteins do membranes have?

A

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

143
Q

What is a leaflet?

A

Half of a phospholipid bilayer

144
Q

How can we visualise the movements of proteins in the bilayer?

A

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

145
Q

How do proteins provide functional asymmetry of membranes?

A

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

146
Q

What are the three types of membrane proteins?

A
  • integral (intrinsic)
  • peripheral (extrinsic)
  • lipid anchored
147
Q

What are integral membrane proteins?

A

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

148
Q

What are peripheral membrane proteins?

A

Interact with membrane via polar heads of integral proteins

149
Q

What are lipid-anchored membrane proteins?

A

Protein polypeptide remains in aqueous phase

Can be fatty acid anchored, isoprenoid anchored or glycosylphosphatidyl-inositol anchored

150
Q

What are the two classes of membrane transfer proteins?

A

Channels/pores - central passage for ions/molecules
Transporters - passive and active

151
Q

What are the transporters transfer process?

A

Uniport - single solute
Symport - same direction
Antiport - opposite direction

152
Q

What are the two types of active transport?

A

Primary - coupled to energy source
Secondary - coupled to ion conc gradient created by primary

153
Q

What is ModABC?

A

ATP-binding cassette transporter

154
Q

What is the work equation?

A

work (J) = force x distance

e.g. dropping apple from string

Starts at rest then moves in the direction needed to reduce potential energy

155
Q

What is pico?

A

x 10-12

e.g. piconeutons per nanometre

156
Q

What is kinetic energy?

A

KE = 1/2mv2

Energy is conserved

If a ball bangs into another - kinetic energy remains the same before and after

157
Q

What is the Boltzmann distribution?

A

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

158
Q

What is KbT?

A

Typically the energy of one molecule

Boltzmann constant (Kb) x T (temp)

Units = JK-1

To find energy per moles - do RT (gas constant)

159
Q

Is PE the same from folded to unfolded?

A

Yes!
e.g. if increased by 10 when folded, decreased by 10 when unfolded

Change of potential energy = change of enthalpy

160
Q

Does an unfolded or folded protein have more entropy?

A

Unfolded - at higher temperatures get more probable - still not as likely as folded

161
Q

How is free energy useful?

A

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

162
Q

What is the entropy of mixing?

A

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

162
Q

What is the free energy for transferring one molecule from a to b?

A

delta G = kb x T x ln (Cb/Ca)

in moles: kb changed to R (gas constant)

If Cb = Ca then ln = 0

163
Q

How do chemical reactions and free energy relate?

A

For a reaction to go forward - delta g for reactants must be higher than products (Forward < backward reaction)

164
Q

What is the equilibrium constant?

A

Keq = [product] eq x [product]eq/ [reactant]eq x [reactant]eq

  • fixed number
165
Q

What is the mass action ratio?

A

r = [product][product]/[reactant][reactant]

  • depends on actual conc

The difference between mass action ratio and Keq shows how far we are from equilibrium

166
Q

What does delta G depend on?

A

Concentration independent terms

Concentration dependent terms - entropic

167
Q

What are the features of delta g?

A

Free energy depends on concs
What at equilibrium = 0
Delta G values in tables are arbitrary - depend on conditions
Delta G will change for different sets of conditions

168
Q

What is delta g ^o

A

Standard state - concs are all 1M - usually pH = 7 so we can ignore [h]

delta g = delta g^o + RT([P]/[R])

169
Q

How do we measure delta g^o

A

We let reaction reach equilibrium

0 = delta g ^o + RTlnk
delta g^o = -RTlnK

170
Q

How do we calculate energy change for ions moving across a membrane?

A

= 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

171
Q

What do reaction rates depend on?

A

Temperature
Pressure
pH
Ionic strength
Reagent conc

172
Q

What is the irreversible unimolecular kinetic model?

A

A -> B
not reversible

Rate is proportional to concentration of A

v = -delta A/deltaT = deltaB/deltaT

rate = k[A] - FIRST ORDER

173
Q

What is first order vs unimolecular?

A

First order - rates directly proportion to concentration
Unimolecular - A –> B

174
Q

What are the units for k?

A

first order - usually min-1 or s-1
second - usually conc-1time-1

175
Q

What is the reversible unimolecular kinetic model?

A

Rate of formation of B = k1[A] - k2[B]

176
Q

What is the irreversible bimolecular kinetic model?

A

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

177
Q

What is the average time a molecule stays in state a?

A

Average time = 1/k1

Same for B = 1/k2

178
Q

What is Keq in relation to k1 and k2?

A

Keq = k1/k2

179
Q

What is Keq when equilibrium shifts?

A

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

180
Q

What is a way to work out the proportion of B?

A

[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

181
Q

What is a bimolecular reversible reaction?

A

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

182
Q

What is KD?

A

KD = koff/kon

can be [P][L]/[PL] or K-1/K1

Describes ligand binding

Stronger binding = lower KD

183
Q

What is the fraction of bound protein?

A

[P][L]/[PL] = Kd

[PL]/[P]total = [L]/[L] + Kd

[P] total = [P] + [PL]
[L] total = [L] + [PL]

184
Q

What are the rates of a bimolecular reversible reaction at equilibrium?

A

Rates equal

k1[P][L] = k-1[PL]

We write equilibrium as dissociation: PL -><- P + L

185
Q

How does the fraction of [PL] change as [L] changes?

A

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

186
Q

What is the binding curve of [L] and fraction of [P] bound?

A

[PL]/[P]total on y axis
[L] on x axis

graph increases then plateaus - saturation point

187
Q

What does a concentration-rate graph of a simple enzyme look like?

A

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

188
Q

What is the Michaelis Menten equation?

A

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

189
Q

What is kcat?

A

first order rate constant describing the reaction of the ES complex to give products

ES –> E + P

190
Q

What does kcat/KM mean?

A

Second order rate constant describing the reaction of E + S to give enzyme and products

191
Q

What is KM?

A

The michaelis constant - the conc of substrate which produces half maximal rate - 1/2 vmax

192
Q

Do simple enzymes have two steps?

A

Yes!

E + S –><– ES —> E + P

They reversibly make a complex ES which irreversibly makes a product

193
Q

What is a competitive inhibitor and how does it affect Km and Kcat?

A

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
194
Q

What is an allosteric inhibitor?

A

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

195
Q

How can we measure enzyme activity?

A

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)

196
Q

What is arylsulphatase?

A

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

197
Q

What happens when [S] is really small compared to Km?

A

Km + [S] = km

Vmax[S]/Km

Kcat/Km x [S] x [E]T - second order

198
Q

What is the double reciprocal Menton graph?

A

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

199
Q
A

Y = kc + C

200
Q

What makes a good signal?

A

Unique enough to relay a defined signal and only detected by correct receptors

Synthesised, released or altered quickly

Degraded quickly

201
Q

What signalling do first messengers do?

A

Endocrine (hormones)
Paracrine (local mediators)

202
Q

What are examples of endocrine signalling?

A

Adrenaline - aa derived - increases BP, HR and metabolism

Insulin - protein

Testosterone/oestrogen - steroid

203
Q

What are examples of paracrine signalling?

A

Histamine - from mast cells - aa derived

ACh - from nerve terminals

204
Q

How to cells receive many signals?

A

All receptors are proteins - 3D shape enable specificity

Affinity is mediated by non-covalent bonds

We want high affinity and high specificity

205
Q

What is receptor activation and signalling?

A

Transmembrane protein - ligand binding causes conformative change - receptor is then activated and changes signal from extra to intracellular

206
Q

What are the different classes of receptors?

A

Ligand gated
G-protein coupled
Enzymic

207
Q

What is a ligand gated ion channel?

A

A multi-subunit pore where specific ions can pass - usually in synapses

208
Q

What is the acetylcholine receptor?

A

A sodium channel - when bound subunits rotate to open pore

Causes contraction, learning/addiction (nicotine)

209
Q

What are protein kinases?

A

The phosphorylation of AA side groups affects enzyme activity

Kinases can activate other kinases

210
Q

What is receptor tyrosine kinase signalling?

A

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

211
Q

What is Ras GTPase switch?

A

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)

212
Q

Why is Ras important?

A

Important oncogene - some mutations lock Ras in the on state which can cause cancer

213
Q

What causes calcium release?

A

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

214
Q

What are G-protein-coupled receptors?

A

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

215
Q

Do GPCRs produce cAMP?

A

Yes!

Adenylyl cyclase produces cAMP

cAMP activates protein kinases - can boost energy release

216
Q

What do receptors activate?

A
  • cellular metabolism
  • transcription
  • cell division
  • changes in cytoskeleton
217
Q

What is the first law of thermodynamics?

A

Energy is conserved - it can neither be created nor destroyed

218
Q

What does life require?

A

Negative entropy

219
Q

What is catabolism and anabolism?

A

Anabolism = making stuff (endergonic - non-spontaneous - positive gibbs)

Catabolism = breaking stuff (exergonic - spontaneous - negative gibbs)

Catabolism provides the energy and precursors for anabolism

220
Q

What is the difference between exergonic and endergonic?

A

Endergonic - requires the input of energy (anabolism)
Exergonic - releases energy (catabolism)

221
Q

What are metabolic pathways?

A

Series of reactions - usually needing enzymes that catalyses the conversion of one molecule to another

222
Q

What must a metabolic pathway be?

A
  • physically possible
  • thermodynamically likely (- gibbs)
  • kinetically feasible
  • shielded from unwanted side reactions
223
Q

What are the benefits of metabolic pathways?

A
  • 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)
224
Q

What are common features of metabolism?

A
  • many common pathways
  • 6 basic types of reaction
  • common organisation patterns
  • common regulatory principles
  • common co-factors
  • use ATP
225
Q

What are the 6 basic types of reaction?

A

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

226
Q

What are the common organisational paradigms (patterns)?

A

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)

227
Q

What are the benefits of compartmentalisation?

A
  • 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
228
Q

How are metabolic pathways studied?

A
  • cell fractionation
  • inhibitors
  • radiolabelling
  • mutants
229
Q

What is cell fractionation?

A

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

230
Q

How can we use inhibitors to study metabolic pathways?

A

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
231
Q

What did radiotracing allow us to do?

A

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

232
Q

How can we study metabolism using mutants?

A

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

233
Q

How do we obtain energy from reduced energy sources?

A

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

234
Q

What atom is reduced in carbon dioxide?

A

Oxygen, carbon is oxidised

This is because the electrons are unevenly shared

In methane - carbon is reduced

235
Q

What bonds between C-H, C-O and C–O is least stable?

A

C-H so energy is released when replaced by C-O or C–O

236
Q

What happens to the free energy released during oxidation?

A

Coupled to the generation of activated carrier molecules - can drive endergonic reactions

237
Q

What are some common activated carrier molecules?

A

ATP - phosphate
NADPH/NADH - hydrogen and e
Carboxylated biotin - carboxyl group
S-adenosylmethionine - methyl group
Uridine diphosphate glucose - glucose

237
Q

What is more stable: ATP or ADP and pi?

A

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

237
Q

What is the free energy change of ATP?

A

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

237
Q

Are co-factors kinetically stable?

A

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

238
Q

Is free energy coupled in metabolism?

A

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

238
Q

What is glycolysis?

A

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?

238
Q

What is glycolysis step one?

A

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

239
Q

How does hexokinase phosphorylate glucose?

A

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

240
Q

What is the free energy of a coupled reaction?

A

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

241
Q

How does coupling effect the equilibrium constant?

A

The delta G has changed

K = e^ (delta g/RT)

242
Q

What is the second step of glycolysis?

A

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

243
Q

What is the third step of glycolysis?

A

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

244
Q

What is flux in ATP?

A

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

245
Q

What regulates glycolysis?

A

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

246
Q

What is step 4 of glycolysis?

A

Fructose-1,6-bisphosphate is cleaves by aldolase to form dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate (GAP)

Only GAP can go through glycolysis

247
Q

What is step 5 of glycolysis?

A

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

248
Q

What is TIM?

A

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

need to know the mechanism?

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

249
Q

What are the products of glycolysis?

A

2 x pyruvate
2 x ATP
2 x NADH

250
Q

What is step 6 of glycolysis?

A

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

251
Q

What is NADH?

A

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

252
Q

What is step 7 of glycolysis?

A

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

253
Q

What is step 8 of glycolysis?

A

Phosphoglycerate mutase transfers phosphoester linkage from carbon 3 to 2

Forms 2-phosphoglycerate

254
Q

What is step 9 of glycolysis?

A

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

255
Q

What is step 10 in glycolysis?

A

Pyruvate kinase transfers a phosphate to ADP and forms pyruvate from phosphoenolpyruvate

This reaction is far from equilibrium so pyruvate conc is kept low

256
Q

What happens after glycolysis?

A

O2: converted to acetyl-coA via link reaction

No O2: pyruvate fermented to lactate

257
Q

What is fermentation?

A

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)

258
Q

What enzymes drive the big energy changes in glycolysis?

A

Hexokinase
Phosphofructokinase
Pyruvate kinase

259
Q

What is a redox potential?

A

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)

260
Q

How are electrons transported in the ETC?

A

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

261
Q

What are some inhibitors of the ETC?

A

Complex I - barbitone
Complex III - antimycin
Complex II - malonate
Complex IV - azide

262
Q

What happens to pyruvate after glycolysis in presence on O2?

A

Will be converted to Acetyl Co-enzyme A via link reaction

Acetyl CoA is metabolised in the kreb cycle

These reactions form CO2

263
Q

What is the link reaction?

A

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

264
Q

What is pyruvate dehydrogenase?

A

Enzyme complex in link reaction

Binds 5 cofactors:
- thiamine pyrophosphate
- lipoamide
- FAD/FADH2
- CoA
- NAD/NADH

265
Q

What is CoASH?

A

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

266
Q

What is FAD/FADH2?

A

Flavin adenine di-nucleotide

Common activated carrier molecule capable of carrying 2 electrons and 2 protons

267
Q

What is pyruvate dehydrogenase?

A

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

268
Q

What is the first step of the kreb cycle?

A

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

269
Q

What is step 2 of kreb cycle?

A

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

270
Q

What is step 3 of kreb cycle?

A

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

271
Q

Why is CO2 important?

A

Decarboxylation gives a strong thermodynamic pull to a reaction as:

  • very stable
  • easily escapes (soluble)
  • more products than reactants so positive entropy
272
Q

What is step 4 of the Kreb cycle?

A

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

273
Q

What is step 5 of kreb cycle?

A

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

274
Q

How does succinyl-CoA synthetase work?

A

CoA is displaced by phosphate - succinyl phosphate.

Histidine then removes Pi forming succinate and phosphohistidine - phosphate then transferred to ADP - ATP (substrate level phosphorylation)

275
Q

What is step 6 of kreb cycle?

A

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+

276
Q

What is step 7 of kreb cycle?

A

Fumarase converts fumarate to malate by adding water across c=c bond

277
Q

What is step 8 of kreb cycle?

A

Malate dehydrogenase uses NAD+ to converts OH on malate to a carbonyl group

Makes oxaloacetate

278
Q

What is an amphibolic cycle?

A

Catabolic and anabolic

e.g. kreb cycle

279
Q

How was kreb cycle uncovered?

A

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

Adding any poison caused succinate accumulation - must be a cycle

280
Q

What is the warburg manometer?

A

Measures changed in pressure caused by oxygen uptake

CO2 is absorbed by filter paper soaked in KOH

Substrate added by side flask

281
Q

How can we make acetyl CoA from fats?

A

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 are transported into mitochondria

In mitochondrial matrix

Form Acetyl CoA and NADH/FADH2 (can be used in oxid phos)

282
Q

What are the electron donors in the ETC?

A

NADH (complex I) and FADH2 (complex II - succinate?) 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

283
Q

How can we study the mitochondria?

A

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

284
Q
A
285
Q

How do electrons flow in ETC?

A

Through redox reactions - redox potentials

286
Q

What are redox potentials?

A

Measure of the affinity of a redox couple for electrons

More negative = more likely to donate
More positive = more likely to accept

287
Q

What is chemiosmotic coupling?

A

Free energy released as electrons travel from negative to positive redox potentials - used to move protons from matrix to IMS (low to high)

288
Q

What is the free energy in the redox reaction in the ETC?

A

Downhill (-ve delta g) - energy used to couple to proton transport

289
Q

What is a standard redox potential?

A

Measured using the compound with a standard hydrogen half cell containing 10-7 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

290
Q

What are some redox potentials in the ETC?

A

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

291
Q

How do we work out delta g from redox potentials?

A

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

292
Q

How do we calculate the actual redox potential?

A

Em = Em0 + (RT/nF) ln([ox]/[red])

n = number of e transfered

CHANGE mV to V

293
Q
A
293
Q

What is succinate dehydrogenase?

A

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

293
Q

What is ubiquinone?

A

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

293
Q

Which complexes pump H+ into the IMS?

A

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

294
Q

What else feed UQ?

A

FADH2 from fatty acid B oxidation (fatty acid CoA dehydrogenase) and NADH from glycolysis (glycerol 3-phosphate dehydrogenase)

295
Q

What is cytochrome bc1?

A

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)

296
Q

How do complex I & II interact with III?

A

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

297
Q

What is the structure of cytochrome c?

A

A small soluble electron carrier

In intermembrane space

Each cytochrome c binds to one electron which reduces Fe3+ to Fe2+

298
Q

What is complex IV?

A

Transfers electrons from cyt c to O2 - needs 2 electrons and 2 hydrogen ions - makes h+

The free energy pumps 2 H+ in

299
Q

What is complex V?

A

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

300
Q

How do we calculate the free energy of the PMF?

A

pmf = membrane potential (trident) - 2.3(RT/F x (change in pH))

Delta g = zF(pmf)

301
Q

What is the efficiency of forming ATP by pmf?

A

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%

302
Q

How many H+ are transferred per cofactor?

A

For each NADH = 10H+
For each FADH2 = 6H+

2.5 ATP per NADH
1.5 ATP per FADH2

303
Q

How many ATP do we make per glucose?

A

30

304
Q

What is the chemiosmotic hypothesis?

A

Peter Mitchell suggested that the electrochemical proton gradient generated by electron transport was used to generate ATP

305
Q

What is evidence for chemiosmosis?

A
  • 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 (allowed proton diffusion)
  • when adding a proton pump in mitochondria instead of ATP synthase caused ATP - showed there was no high energy intermediates
306
Q

How does Γ and k effect delta g?

A

Γ = product conc
k = equilibrium product conc

Γ&laquo_space;K and Γ/K <1 then delta g is more negative

Γ» K and Γ/K >1 then delta g is more positive

307
Q

What does a low pka mean?

A

Strong acid - wants to give away proton