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Biomed > Biochemistry S1 Y1 > Flashcards

Biochemistry S1 Y1 Flashcards

1
Q

What do the reactions in living systems obey?

A

Thermodynamic laws

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2
Q
  • What is thermodynamics?
  • What can make a reaction more thermodynamically favourable?
A
  • The direction of a process
  • Coupling of reactions
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3
Q

What is kinetics?

A

The rate of a process

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4
Q

What is a system?

A

A part of the universe

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5
Q

What are the surroundings?

A

The rest of the universe outside of a system that interacts with a system

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6
Q

What is a boundary?

A

The barrier between the system and the surroundings

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7
Q

What is a process?

A

Any change in a system

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8
Q

What are the two main types of energy transfer?

A
  1. Heat (random motion = energy transfer)
  2. Work (organised motion = energy transfer)
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9
Q

Difference between open and closed system?

A

Open has matter and energy in and out, closed only has energy in and out

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10
Q
  • What is the first law of thermodynamics?
  • What does it imply?
A
  • “Energy is never created or destroyed but it can be changed to another form or be transported elsewhere”
  • Reaction can occur both forward and backwards and deals with energy balance
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11
Q

What is a state function?

A

Something that only depends on the current state of a system e.g. energy

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12
Q

What is meant by enthalpy change?

A

The heat change of a reaction when pressure is constant (it also reflects the number and types of chemical bonds present)

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13
Q

Equation for enthalpy change (involving internal energy)?

A

ΔH= ΔU+pΔV

Enthalpy change = change in internal energy + (pressure x change in volume)

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14
Q

What is the second law of thermodynamics?

A

Spontaneous change in a system increases the combined entropy of the system and surroundings

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15
Q

What does Gibbs free energy (G) measure?

A

Driving force of process

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16
Q
  • Equation for free energy change?
  • Units?
  • What is ΔG?
A
  • ΔG = ΔH – TΔS
  • Units:
    ΔG and ΔH in J mol^-1 or kJ mol^-1
    T in K
    ΔS in J mol^-1 K^-1
  • Change from free energy of reactants to free energy of products
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17
Q

What will a system far from equilibrium experience to return to stable equilibrium?

A

An irreversible process

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18
Q

ΔG > 0 is …? (what does this mean)

A

ENDERGONIC (forward unfavourable, reverse spontaneous)

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19
Q

ΔG = 0 is …? (what does this mean)

A

EQUILIBRIUM (no change)

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20
Q

ΔG < 0 is …? (what does this mean)

A

EXERGONIC (forward favourable and spontaneous)

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21
Q

Entropy equation?

A

S = K x ln x w
Entropy = Boltzmann constant x natural log x number of ways of arranging system

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22
Q

What is the equation for ΔG relating to equations like aA + bB –> cC + dD?

A

ΔG = ΔG° x R x T x loge x [products]/[reactants]

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23
Q
  • What does ΔG° show?
  • ΔG°’?
A
  • All reactants at 1M concentration
  • pH=7
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24
Q

Equation to find ΔG°?

A

ΔG° = -RT ln Keq (eq is subscript)
(ΔG = 0 and Keq is found by [products]/[reactants])

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25
Q

If ΔG° is…, Keq is…
1. ΔG° < 0
2. ΔG° = 0
3. ΔG° > 0

A
  1. Keq > 1
  2. Keq = 1
  3. Keq < 1
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26
Q

6 reasons why ATP is the major energy currency?

A
  1. The highly charged phosphate group make as a negative ΔG of ATP hydrolysis
  2. Electrostatic repulsion is relieved when last phosphate bond is hydrolysed
  3. Inorganic phosphate stabilises as a resonance hybrid
  4. Thermodynamically unstable but kinetically stable
  5. High activation energy for ATP hydrolysis (not hydrolysed before use)
  6. Cell can regulate energy distribution carried out by ATP by regulating the enzymes that work on it
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27
Q

5 aliphatic amino acids?

A

Alanine (Ala, A)
Valine (Val, V)
Leucine (Leu, L)
Isoleucine (Ile, I)
Methionine (Met, M)
all are hydrophobic and drive folding

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28
Q

3 aromatic amino acids?

A

Phenylalanine (Phe, F)
Tyrosine (Tyr, Y)
Tryptophan (Trp, W) (has 2 phenyl rings, others have 1)

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29
Q

2 acidic amino acids?

A

Aspartic acid (Asp, D)
Glutamic acid (Glu, E)

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30
Q

3 basic amino acids?

A

Histidine (His, H)
Lysine (Lys, K)
Arginine (Arg, R)

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31
Q

5 polar amino acids?

A

Serine (Ser, S)
Threonine (Thr, T)
Cysteine (Cys, C)
Asparagine (Asn, N)
Glutamine (Gln, Q)

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32
Q

2 unique amino acids?

A

Glycine (Gly, G) - flexible, creates many conformations
Proline (Pro, P) - creates kink in chain

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33
Q

PRACTICE USING CHEMDRAW TO DRAW PEPTIDES

A
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34
Q

Light absorption in amino acids:
- Amino acids that can?
- What can it be used for?
- Equation used?

A
  • Aromatics
  • Measuring protein concentration
  • Beer-lambert law
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35
Q

Ionisation in amino acids:
- What occurs at low pH?
- As pH increases?
- What is an amino acid like at high pH?
- What is the isoelectric point?

A
  • Amine is protonated (H3N+)
  • Carboxylic acid is deprotonated (COO-)
  • Amine normal (NH2), carboxylic acid is deprotonated
  • The point of no overall electric charge (zwitterion)
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36
Q

Chirality in amino acids:
- What are chiral stereoisomers?
- Why do enzymes bind to one of the D or L stereoisomers but not the other?

A
  • Molecules with the same bonds but a different spatial arrangement of them to form non-superimposable mirror images
  • They have a different shape, so the enzyme is only specified to one
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37
Q

General equation for weak acid?

A

HA + H2O <–> H3O+ + A-

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38
Q

How can you tell the difference between whether a molecule is a L or D stereoisomer?

A

If you go clockwise around the molecule:
CORN (COOH - R group - NH2) = L
CONR = D

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39
Q

Which end of a polypeptide is classed as the beginning?

A

The amino end

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40
Q

How does the R - S system of classification of amino acids work?

A

If priority 1-4 is:
Clockwise = R
Anticlockwise = S

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41
Q
  • What does the double bond between N and C in peptides prevent?
  • Does a cis or trans configuration minimise steric clashes the most?
A
  • Rotation
  • Trans (R groups on opposite faces so are as far away as possible - but X-Pro linkages have steric clashes cis or trans)
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42
Q

What are the two types of rotation about bonds in a polypeptide?

A
  • Phi = bond between N and alpha-carbon
  • Psi = bond between alpha-carbon and carbonyl carbon
    BUT, only 1/4 of Phi and Psi configurations are possible as steric clashes are very high
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43
Q
  • What stabilises alpha helix?
  • 2 types of alpha helix?
  • What is their dipole moment and what does it allow?
A
  • H-bonds
  • Clockwise (most common) and anticlockwise
  • Positive amino end and negative carbonyl end. Allows binding of negatively charged molecules
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44
Q

Beta-pleated sheets:
- How are polypeptide chains joined?
- 2 types and how they differ?

A
  • H bonds between extended Beta sheets
  • Antiparallel (H bonds connect each amino acid to a single amino acid on other chain) OR parallel (one amino acid bound to 2 amino acids on other chain)
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45
Q

Beta loops:
- What do they allow?
- 2 types and how they differ?

A
  • Reversals in polypeptide chain and 1st and 4th amino acids form H bond
  • Type I = AA, proline, AA, AA (proline allows kink)
    Type II = AA, AA, glycine, AA (AA = amino acid)
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46
Q

What does amphipathic mean?

A

Protein folding to create inner hydrophobic, non-polar core to exclude hydrophobic residues from water

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47
Q

What is tertiary structure controlled by?

A

Interactions of side-chains (disulfide bridges, ionic bridges, H bonds, hydrophobic interactions)

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48
Q

What are disulfide bridges reduced to?

A

SH groups

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49
Q

How is the thermodynamically favoured structure of active ribonuclease found?

A

Native disulfide pairings stabilise it by breaking and reforming until the protein folds into the preferred structure due to a decrease in free energy

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50
Q

What are the 5 steps of the life cycle of influenza neuraminidase?

A
  1. Flu particle binds to sialic acid on proteins on cell membrane
  2. Endocytosis
  3. Viral Replication
  4. Budding
  5. Release
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51
Q

Structure of neuraminidase?

A

Tetrameric (alpha helices and beta ribbons)

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52
Q

How is flu treated?

A

Competitive inhibitor of neuraminidase prevents it from binding to sialic acid

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53
Q

What is the proteome?

A

Entire complement of proteins expressed in a cell OR set of proteins expressed under specific conditions

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54
Q

4 post-translational modifications?

A
  1. Addition of small chemical groups
  2. Addition of small proteins
  3. Addition of complex molecules
  4. Amino acid processing
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55
Q

8 steps of protein purification?

A
  1. Transformation (plasmid into E. coli)
  2. Selection (of colonies)
  3. Cell growth and protein production
  4. Cell lysis
  5. Column chromotography
  6. Dialysis
  7. SDS-PAGE (electrophoresis to check protein purity - only single protein should show)
  8. Protein assay (checks activity - should not have changed)
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56
Q

3 types of column chromatography?

A

Gel filtration, ion exchange, affinity

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57
Q

Gel filtration chromatography:
- How are proteins separated?

A

Separated based on molecular size whereby gel beads trap small molecules so big ones move through and are separated

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58
Q

Ion exchange chromatography:
- How are proteins separated?

A

Separated based on charge whereby negatively charged gel beads bind to positive proteins so negative proteins move through

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59
Q

Affinity chromatography:
- How are proteins separated?

A

Separated based on specific binding interactions whereby antibodies recognise a protein and bind so all the others run out and then the column is washed and all the proteins unbind and are obtained

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60
Q

What do enzymes increase?

A

Rate of chemical transformation

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61
Q

How do proteases degrade proteins?

A

Cleave peptide bonds by hydrolysis

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62
Q

6 classes of enzymes?

A

Oxidoreductases, transferases, hydrolases, lyases, isomerases, ligases

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63
Q
  • Role of oxidoreductases?
  • E.g.?
A
  • Catalyse redox reactions and transfer H and O atoms between substrates
  • Alcohol dehydrogenase
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64
Q
  • Role of transferases?
  • E.g.?
A
  • Transfer functional groups between donors and acceptors (compounds)
  • Hexokinase
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65
Q
  • Role of hydrolases?
  • E.g.?
A
  • Catalyse hydrolysis of substrates (hydrolytic cleavage of C-O, C-N, C-C)
  • Carboxypeptidase A
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66
Q
  • Role of lyases?
A
  • Catalyse removal of a group by means other than hydrolysis or oxidation (e.g. elimanation) and catalyse addition to double bonds
  • Pyruvate decarboxylase
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67
Q
  • Role of isomerases?
  • E.g.?
A
  • Catalyse inter-molecular rearrangement (geometric and structural changes) and addition to double bonds
  • Maleate isomerase
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68
Q
  • Role of ligases?
  • E.g.?
A
  • Catalyse union of two molecules (e.g. C-S, C-O, C-N bonds) using chemical energy
  • Glutamine synthetase
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69
Q

Why does enzyme specificity arise?

A

Precise interaction between enzyme and substrate and the intricate structure of enzyme

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70
Q

What does ΔG determine?

A

If a reaction is spontaneous

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71
Q

What will the concentration of reactants and products always reach?

A

Equilibrium position

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72
Q

How do enzymes use the transition state (S‡) of a substrate to work?

A

They lower the activation energy of the transition state (which is between the substrate forming the product) by using binding energy to stabilise the intermediate

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73
Q

What are enzymes complimentary to?

A

The transition state (NOT substrate)

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74
Q

2 factors of why enzymes can generate large rate?

A
  1. Non-covalent interactions between enzyme and substrate in ES complex (weak interactions+small energy release = stabilised interaction)
  2. Rearrangement of covalent bonds (as water is not a strong enough nucleophile to hydrolyse peptide bonds, proteases allow rearrangement of bonds and catalytic triads can transform molecules and split it into its groups)
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75
Q

4 ways binding energy contributes to catalysis?

A
  1. Enzyme holds reactants close in right orientation (proximity effect - closer = greater rate, orientation effect - reaction always occurs at right orientation)
  2. Weak bonds between enzyme and substrate = desolvation of substrate (water molecules blocking active site removed)
  3. Binding energy in transition states compensates for thermodynamically unfavourable ΔG associated with substrate distortion
  4. Enzyme undergoes conformational change for induced fit
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76
Q

What do enzymes have different levels of?

A

Specificity (involving geometric specificity and stereospecificity)

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77
Q

What does increasing [S] increase?

A

Amount of product until equilibrium

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78
Q

What is V0?

A

Initial rate of catalysis

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79
Q

What is V0 like at low [S]?

A

Increases linearly as [S] increases

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80
Q

What is V0 like at high [S]?

A

It plateaus

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81
Q

What does the michaelis-menten equation link?

A

Enzyme rate of reaction and [S]

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82
Q

What are the two parts of the steady state assumption?

A

First = pre-steady state (too short for observation)
Second = steady state ([ES] steady)

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83
Q
  • V0 equation?
  • Variations?
A

V0 = Vmax x [S]/[S]+Km

  • If [S] &laquo_space;Km then V0=(Vmax x [S])/Km AND enzyme rate is less than Kcat AND [E] = [Et]
  • If [S]&raquo_space; Km then V0=Vmax AND rate of catalysis = Vmax
  • If [S] = Km then V0=Vmax/2
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84
Q

How does Km vary?

A

Enzymes have different Km values for substrates - low Km = better enzyme processing of substrate

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85
Q

What does Km depend on?

A

Substrate, pH, temperature

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86
Q

What does Km = [S] mean?

A

Half of active sites full

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87
Q

What is K1, K2 and K-1?

A

K1 = E + S –> ES
K2 = ES –> E + P
K-1 = ES –> E + S

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88
Q

Km equation?

A

Km = K-1 + K2 / K1

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89
Q

What does K-1&raquo_space; K2 mean?

A

ES dissociates to E + S quicker than product is formed

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90
Q

What does high or low Km mean?

A

High = weak binding
Low = strong binding

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91
Q

What is the turnover number of an enzyme?

A

Number of substrate molecules converted into product by an enzyme molecule in an unit time when enzyme is fully saturated

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92
Q

Equation for Vmax?

A

Vmax = K2 [Et]
WHICH IS Vmax = Kcat [Et]
([Et] is total enzyme conc.)

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93
Q
  • What is Kcat?
  • Equation?
A
  • Turnover
  • Kcat = Vmax / [Et]
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94
Q

What is Kcat / Km a measure of?

A

Catalytic efficiency

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95
Q

What is a major way enzyme activity is controlled?

A

Binding of small molecules/ions inhibits it
(irreversible inhibitors are tightly bound and dissociated slowly, reversible inhibitors rapidly dissociate)

96
Q
  • What are inhibitors?
  • 2 types?
A
  • Molecules that interfere with enzyme catalysis and slow/halt enzymatic transformations
  • Specific and non-specific
97
Q

2 types of specific enzymes?

A
  • Reversible (competitive, uncompetitive and non-competitive)
  • Irreversible
98
Q

How are non-specific enzymes characterised?

A

Inhibit many enzymes (e.g. acids do this)

99
Q

Competitive inhibition:
- Main takeaway?
- What is the mechanism?
- Michaelis-menten equation for this?

A
  • Vmax does not change, Km increases
  • Compete with substrate for active site but do not inactivate the enzyme
  • V0 = Vmax x [S] / αKm + [S]
    α = 1+ [I] / Ki
100
Q

Competitive inhibition:
- What is the equation for the dissociation constant (Ki)?
- What does a smaller Ki mean?
- What can competitive inhibition be overcome with?

A
  • Ki = [E][I] / [EI]
  • More potent inhibition
  • High substrate concentration (reaches same Vmax) as if [S]&raquo_space; [I] then Vmax is still attained
101
Q

Uncompetitive inhibition:
- Main takeaway?
- Mechanism?
- Michaelis-menten equation for this?

A
  • Vmax decreases, Km decreases
  • Inhibitor binds to different site than active site, but only binds to ES complex (does not inactivate enzyme, but does remove fraction of enzyme from reaction)
  • V0 = Vmax x [S] / Km + α’[S]
    α’ = 1 + [I] / Ki’
102
Q

Uncompetitive inhibition:
- How do uncompetitive inhibitors affect Km?
- Where does the inhibitor bind?

A
  • Lower it by lowering [ES] and increasing [ESI] which increases K1
  • ES complex to form ESI complex so no product is formed
103
Q

Non-competitive inhibition:
- Main takeaway?
- Mechanism?
- What is Ki’?
- Michaelis-menten equation for this?

A
  • Vmax decreases, Km stays the same
  • Inhibitors bind to different site than substrate but does not inactivate enzyme
  • ES + I –> ESI
    Ki’ = [ES][I] / [ESI]
  • V0 = Vmax x [S] / αKm + α’[S]
    α = 1 + [I] / Ki
    α’ = 1 + [I] / K’i
104
Q

Non-competitive inhibition:
- What is Vmax reduced to?
- What does not change?
- Equation for Vapp max?
- Why is it not overcome by higher [substrate]?

A
  • Vapp max
  • Km
  • Vapp max=Vmax/(Inhibitor+([Inhibitor / Ki))
  • The concentration of functional enzymes is lowered (Vmax cannot be reached)
105
Q

How can the mechanism of inhibition be determined?

A

Lineweaver-Burke plots (in notes and pp)
e.g. if you increase [substrate] and Km increases, it is competitive

106
Q

Irreversible inhibition:
- Mechanism?
- How do mechanism-based inhibitors work?
- Example?

A
  • The inhibitors covalently modify a functional group which is essential for an enzyme’s function
  • Hijack normal enzyme reaction mechanism and they can be specific to a single protease
  • DIFP inhibits ALL serine proteases
107
Q
  • What does regulating activity of enzyme allow?
  • 4 ways?
A
  • Turning on, turning off or modulating activity of various metabolic pathways
    1. Allosteric enzyme regulation
    2. Reversible covalent enzyme modification?
    3. Proteolytic cleavage
    4. Feedback regulation
108
Q

What is allosteric enzyme regulation?

A

Allosteric modulators/effectors bind and activate or deactivate by changing active site shape

109
Q

What is reversible covalent enzyme modification?

A

Modification of residues (post-translational) by adenylation, methylation, phosphorylation and ADP-ribosylation

110
Q

What is proteolytic cleavage?

A

Enzymes produced are inactive and called zymogens/pro-enzymes and can be activated by removal of a polypeptide segement by proteolytic cleavage

111
Q

What is feedback regulation?

A

End-product inhibits upstream enzyme to decrease rate of production when critical [S] reached

112
Q

3 things carbohydrates ((CH2O)n) do?

A
  • Structural components
  • Energy source
  • Part of protein structure/function
113
Q

Why is glucose the most common carbohydrate?

A
  • Most stable in solution
114
Q

Why are carbohydrates diverse?

A
  • Multiple chiral and pro-chiral carbons
  • Many branching options
115
Q

2 types of carbohydrates?

A
  • Polyhydroxy aldehydes or polyhydroxy ketones
116
Q

What is:
- N = 1?
- N = 2?
- N = 3?

A
  • Methanal
  • Hydroxyethanal
  • Glyceraldehyde (has a chiral carbon so has enantiomers) or dihydroxyacetone
117
Q

How are simple carbohydrates named?

A
  • N = 3 are trioses, N = 4 are tetroses etc …
  • They are all aldo or keto
118
Q

2 enantiomers of glyceraldehyde?

A

D-glyceraldehyde and L-glyceraldehyde (diagrams in notes from 16/11/23)

119
Q

How can the structure of enantiomers of carbohydrates be shown?

A

Fischer projections (straight line, 4 way cross with chiral carbon at centre)

120
Q

How are enantiomers named?

A

The chiral carbon furthest from oxidised carbon (also known as highest number carbon) determines name e.g. if highest number carbon has an OH group on the left and a H on the other it is an L-enantiomer

121
Q

What does multiple chiral carbons mean?

A

Multiple isomers (2^n possible forms where n is the number of chiral carbons)

122
Q

What are epimers?

A

Isomers that are not mirror images of one another (differ at one carbon)

123
Q
  • What do linear saccarides of usually 5 or more carbons form in water?
  • 2 examples?
A
  • Ring structures with an oxygen bridge between carbonyl carbon and another carbon in chain
  • Furanose ring (5 membered, 4C and 1O)
    Pyranose ring (6 membered, 5C and 1O)
    DIAGRAMS IN NOTES 16/11/23
124
Q

Divide of α-glucose vs β-glucose?

A

α-glucose is 1/3 (OH on bottom side)
β-glucose is 2/3 (OH on top side)

125
Q

Why is the structure of glucose not actually flat?

A
  • Six-sided shape should have 120°, but it is actually 109.5° so to reduce strain the structure is not flat - is in chair or boat form (in notes)
126
Q

What is metabolism?

A

Total of all of an organisms’ life sustaining chemical reactions

127
Q

Difference between anabolic and catabolic?

A

Anabolic = small to large molecules (energu requiring)
Catabolic = large broken down to small molecules (energy released)

128
Q

What kind of process is glycolysis?

A

Catabolic reaction in cytosol to extract energy from glucose

129
Q

What is aerobic glycolysis?

A

Glucose –> pyruvate + ATP

130
Q
  • What is anaerobic glycolysis?
  • What depends on this?
A

Pyruvate –> lactate + ATP
- Skeletal muscles

131
Q

Why is most of glycolysis reversible?

A

So gluconeogenesis can occur

132
Q

What are the two phases of glycolysis?

A

Energy-requiring (2 ATP used, glucose phosphorylation) and energy-releasing (2 ATP and 1 NADH per pyruvate)

133
Q

10 steps of glycolysis (diagrams in notes)?

A

PHASE ONE (energy requiring)
1. Uptake and phosphorylation of glucose
2. Isomerisation of glucose-6-phosphate to fructose-6-phosphate
3. Phosphorylation of fructose-6-phosphate to fructose-1,6-bisphosphate
4. Cleavage of fructose-1,6-bisphosphate
5. Interconversion of the triose phosphates

PHASE TWO (energy releasing)
6. Oxidative phosphorylation of GAP to
1,3-biphosphoglycerate
7. Conversion of 1,3-biphosphoglycerate to 3-phosphoglycerate
8. Conversion of 3-phosphoglycerate to
2-phosphoglycerate
9. Dehydration of 2-phosphoglycerate to phosphoenolpyruvate
10. Conversion of phosphoenolpyruvate to pyruvate

134
Q

1 - Uptake and phosphorylation of glucose
- Product?
- Reversible or irreversible?
- 2 enzymes used?

A
  • Glucose-6-phosphate and ADP
  • IRREVERSIBLE
    1. Hexokinase (transfers phosphate from ATP to glucose) and has an Mg2+ cofactor
    2. Glucokinase (type of hexokinase in liver, only in blood at HIGH glucose)
135
Q

2 - Isomerisation of glucose-6-phosphate to fructose-6-phosphate
- Process?
- Reversible or irreversible?
- Enzyme?

A
  • Aldose-ketose isomerisation
  • REVERSIBLE
  • Phosphohexose isomerase (catalyst)
136
Q

3 - Phosphorylation of fructose-6-phosphate to fructose-1,6-bisphosphate
- What is used?
- Reversible or irreversible?
- What is this step also known as?
- Enzyme?

A
  • 1 ATP
  • IRREVERSIBLE (so glucose-6-phosphate does not reform)
  • Bottleneck/committed step of glycolysis
  • Phosphofructokinase-1 enzyme
137
Q

4 - Cleavage of fructose-1,6-bisphosphate
- 2 products?
- Which product can continue in cycle?
- What happens to other product?
- Reversible or irreversible?
- Enzyme?

A
  • Glyceraldehyde-3-phosphate (GAP) and dihydroxyacetone phosphate (DHAP)
  • GAP
  • DHAP is converted into GAP
  • REVERSIBLE
  • Aldolase
138
Q

5 - Interconversion of the triose phosphates
- What occurs?
- Reversible or irreversible?
- Enzyme?

A
  • DHAP –> GAP to stop DHAP wastage
  • REVERSIBLE
  • Triose phosphate isomerase
139
Q

6 - Oxidative phosphorylation of GAP to
1,3-biphosphoglycerate
- What does it yield?
- How does the fate of NADH differ in aerobic and anaerobic conditions?
- Reversible or irreversible?
- Enzyme?

A
  • Energy
  • Aerobic = enters mitochondria, anaerobic = used by lactate dehydrogenase
  • REVERSIBLE
  • Glyceraldehyde-3-phosphate dehydrogenase
140
Q

7 - Conversion of 1,3-biphosphoglycerate to 3-phosphoglycerate
- What is it also known as?
- What forms?
- Reversible or irreversible?
- Enzyme?

A
  • Substrate-level phosphorylation
  • ATP
  • REVERSIBLE
  • Phosphoglycerate kinase and Mg2+
141
Q

8 - Conversion of 3-phosphoglycerate to
2-phosphoglycerate
- Reversible or irreversible?
- Enzyme?

A
  • REVERSIBLE
  • Phosphoglycerate mutase enzyme and Mg2+
142
Q

9 - Dehydration of 2-phosphoglycerate to phosphoenolpyruvate
- What is released?
- What is the product also known as?
- Reversible or irreversible?
- Enzyme?

A
  • Water
  • A high-energy phosphoryl compound called PEP
  • REVERSIBLE
  • Enolase and Mg2+
143
Q

10 - Conversion of phosphoenolpyruvate to pyruvate
- What is this step known as?
- Reversible or irreversible?
- Enzyme?

A
  • Second substrate-level phosphorylation
  • IRREVERSIBLE
  • Pyruvate kinase and Mg2+ and K+
144
Q

Anaerobic glycolysis:
- What does pyruvate to lactate regenerate?
- Reversible or irreversible?
- Enzyme?

A
  • NAD+ from NADH
  • REVERSIBLE
  • Lactate dehydrogenase
145
Q

Overall glycolysis equation?

A

Glucose + 2ATP + NAD+ + 4ADP + 2Pi
–>
2 pyruvate + 4 ATP + 2 NADH + 2H+

146
Q

What is glycogenesis?

A

Making new glycogen

147
Q

What is glycogenolysis?

A

Breakdown of glycogen

148
Q
  • What is gluconeogenesis?
  • Does it require energy?
A
  • Making new glucose from non-glucose substrates like lactate, glycerol and some amino acids (ends with pyruvate to glucose)
  • Yes (endogenous glucose pathway)
149
Q

How are the non-carbohydrate precursors made into glucose (mostly in the liver)?

A
  • Converted to intermediates of glycolysis or pyruvate:
    1. Lactate dehydrogenase converts lactate to pyruvate
    2. Transaminase converts alanine to pyruvate
    3. Glycerol kinase and glycerol phosphate dehydrogenase convert glycerol to DHAP
150
Q

What are the non-equilibrium reactions of glycolysis catalysed by?

A

Hexokinase, phosphofructokinase and pyruvate kinase - reversed by separate and distinct reactions in gluconeogenesis direction

151
Q

3 steps of gluconeogenesis?

A
  1. Pyruvate converted to phosphoenolpyruvate by 2 steps
  2. Fructose-1,6-bisphosphate + H2O –> fructose-6-phosphate + PO3-
  3. Glucose-6-phosphate + H2O –> glucose + PO3-

STEPS INBETWEEN ARE REVERSIBLE SO DO NOT NEED TO BE REVERSED

152
Q

1 - Pyruvate converted to phosphoenolpyruvate by 2 steps
- What are the two steps and the enzymes that are used and where?

A
  1. Pyruvate to oxaloacetate
    Uses pyruvate carboxylase
    In mitochondria
  2. Oxaloacetate to phosphoenolpyruvate
    Uses phosphoenolpyruvate carboxykinase
    In cytoplasm
153
Q

2 - Fructose-1,6-bisphosphate + H2O –> fructose-6-phosphate + PO3-
- Enzyme?
- Where?

A
  • Fructose-1,6-bisphosphatase
  • Cytoplasm
154
Q

3 - Glucose-6-phosphate + H2O –> glucose + PO3-
- Enzyme?
- Where?

A
  • Glucose-6-phosphatase
  • Endoplasmic reticulum
155
Q

Overall equation for gluconeogenesis?

A

2 pyruvate + 6 ATP + 2 NADH –> Glucose + 6Pi + 6 ADP + 2 NAD+

156
Q
  • Pyruvate is converted to oxaloacetate in the mitochondria but why can this not enter the liver and kidneys?
  • How is this overcome?
  • What then happens to oxaloacetate?
A
  • They do not have the receptors
  • Converted to malate by malate dehydrogenase and then converted back to oxaloacetate
  • Converted to PEP (step 2 of gluconeogenesis)
157
Q

5 steps of glucose-6-phosphate into ER and glucose out into cytosol?

A
  1. Transporter protein 1 (T1) transports glucose-6-phosphate in
  2. Stabilising protein (SP) binds Ca2+ required to glucose-6-phosphatase
  3. Glucose-6-phosphatase catalyses glucose-6-phosphate to glucose
  4. Transporter protein 2 (T2) transports Pi into cytosol
  5. Transporter protein 3 (T3) transports glucose into cytosol
158
Q

Difference in a rapid or long-term response to environment by changing rates of metabolic processes?

A

Rapid = change in enzyme activity
Long-term = gene expression/protein synthesis of an enzyme

159
Q

3 things enzymes control?

A
  1. Control of processes
  2. Direction of reaction
  3. Heat of a reaction
160
Q

What is feedback inhibition?

A

Product inhibits reaction

161
Q

What is feedforward regulation?

A

A product is required to produce the final product

162
Q

4 factors for controlling fluxes through glycolysis?

A
  1. Substrate (glucose) availability
  2. Concentration of enzymes (e.g. rate-limiting like hexokinase)
  3. Allosteric regulation of enzymes (binding of small molecules to site different to active site)
  4. Covalent modification of enzymes (e.g. phosphorylation)
163
Q

Substrate availability in glycolysis:
- What regulates glucose uptake?
- How does GLUT 2 work?
- How does GLUT 4 work?

A
  • GLUT family of transporter proteins
    1. GLUT 1 = in RBCs and controls basal
      glucose uptake
    2. GLUT 2 = in liver cells and pancreas β-
      cells and controls glucose uptake and
      removes excess from blood
    3. GLUT 4 = in muscle cells and adipocytes
      to remove excess glucose (regulated by
      insulin)
  • At basal level the correct amount of glucose enters so the correct amount of ATP forms (K+ channels open) BUT above basal level there is too much glucose entering so there is too much ATP (K+ channels close, Ca2+ channels open, insulin vesicles fuse with membrane and insulin is released)
  • Insulin triggers GLUT 4 vesicles to bind with membrane so glucose enters the muscle and blood glucose lowers - when the glucose enters a cell it is converted to glucose-6-phosphate and trapped by hexokinase
164
Q

Enzyme concentration and activity in glycolysis:
- What are the 3 rate limiting enzymes?
- What regulates the concentration?

A
    1. Hexokinase/glucokinase (step 1)
    2. Phosphofructokinase (PFK) (step 3)
    3. Pyruvate kinase (step 10)
  • Hormones (such as insulin that triggers transcriptional activator)
165
Q

Allosteric regulation of enzymes in glycolysis:
- 2 things it can do?
- What inhibits hexokinase?
- What inhibits glucokinase?
- What inhibits and activates phosphofructokinase?
- What inhibits and activates pyruvate kinase?

A
  • Inhibits or activates enzymes
  • Glucose-6-phosphate (product inhibition)
  • RP if blood glucose is <5mM
  • Inhibited by high ATP and citrate
    Activated by low AMP and fructose-2,6-bisphosphate
  • Inhibited by ATP and acetyl-CoA
    Activated by fructose-1,6-bisphosphate (feedforward)
166
Q

Covalent modification of enzymes in glycolysis:
- What enzyme is regulated by this?

A
  • Pyruvate kinase is regulated by phosphorylation and it is less active when it is phosphorylated
167
Q

When there is high blood glucose, pancreatic β-cells release insulin to increase glycolysis, what are the 4 things it triggers to do this?

A
  1. GLUT 4 moves to the surface to allow glucose influx in muscle cells
  2. Increases transcription of hexokinase, phosphofructokinase and pyruvate kinase
  3. Activates PFK2 which increases formation of fructose-2,6-bisphosphate to activate PFK
  4. Activation of pyruvate kinase by the activation of phosphoprotein phosphatase 1 which dephosphorylates pyruvate kinase
168
Q

What is the mechanism of hexokinase regulation?

A

Feedback loops as glucose-6-phosphate is a product inhibitor that increases at low ATP demand

169
Q

What is the regulation of PFK dependent on?

A

The energy status of the cell

170
Q

How is the regulation of glycolysis and gluconeogenesis related?

A

They are said to be reciprocally regulated

171
Q

What are the 4 rate limiting enzymes in gluconeogenesis?

A
  1. Glucose-6-phosphatase (step 1 - converts glucose-6-phosphate to glucose in the ER)
  2. Fructose-1,6-bisphosphatase (step 3)
  3. Pyruvate carboxylase and PEPCK (step 10)
172
Q

4 ways flux through gluconeogenesis is controlled?

A
  1. Substrate (pyruvate and precursors) availability
  2. Concentration of enzymes (e.g. rate-limiting)
  3. Allosteric regulation of enzymes (binding of small molecules to site different to active site)
  4. Covalent modification of enzymes (e.g. phosphorylation)
173
Q

Enzyme concentration in gluconeogenesis:
- Role of glucagon (3)?

A
  1. Down-regulates expression of glycolytic enzymes
  2. Upregulates expression of gluconeogenic enzymes
  3. Stimulates glycogenolysis
174
Q

Allosteric regulation of enzymes in gluconeogenesis:
- Fructose-1,6-bisphosphatase?
- PFK?
- Pyruvate carboxylase?

A
  • Inhibited by metabolites, regulated by energy levels and downstream metabolites like citrate
  • Activated by metabolites
  • Regulated by acetyl-CoA
175
Q

Covalent modification of enzymes in gluconeogenesis:
- How is phosphofructokinase 2 regulated?
- What is the action of PFK2?

A
  • Activated by insulin that triggers phosphorylation at high blood glucose to stimulate glycolysis
  • Regulates glycolysis by converting fructose-6-phosphate to fructose-2,6-bisphosphate by activated fructose-1,6-bisphosphatase at high glucose (reverse at low blood glucose)
176
Q

When there is low blood glucose, pancreatic α-cells release glucagon to increase gluconeogenesis, what are the 2 things it triggers to do this?

A
  1. Increases transcription of gluconeogenic enzymes
  2. Activates fructose-1,6-bisphosphatase which lowers levels of fructose-2,6-bisphosphate which stops inhibition of fructose-1,6-bisphosphatase
177
Q

What does the fate of pyruvate depend on?

A

The conditions

178
Q
  • Fate of pyruvate in anaerobic conditions in a eukaryotic cell?
  • Enzyme?
A
  • Reduced to lactate and regenerates NAD+
  • Lactate dehydrogenase
179
Q
  • Fate of pyruvate in anaerobic conditions in yeast?
  • 2 enzymes?
A
  • Converted to acetaldehyde and then ethanol
  • Pyruvate decarboxylase and alcohol dehydrogenase
180
Q

2 fates of pyruvate in aerobic conditions?

A
  1. Used for synthesis (anabolism)
  2. Oxidised to form CO2 (catabolism)
181
Q

2 steps of aerobic catabolism of pyruvate?

A
  1. Conversion of pyruvate to acetyl-CoA
  2. Oxidation in a cyclical process
182
Q

Pyruvate to acetyl-CoA:
- How?
- Other products?
- Reversible or irreversible?
- Where does it occur?

A
  • COO on pyruvate replaced by CoA
  • CO2 and NADH + H+
  • Irreversible
  • Large multienzyme complex called pyruvate dehydrogenase complex (PDHC/PDH complex)
183
Q

PDHC:
- 3 protein components it is made up of?
- 5 coenzymes it contains?

A
    1. Pyruvate dehydrogenase (E1)
    2. Dihydrolipoyl transferase (E2)
    3. Dihydrolipoyl dehydrogenase (E3)
    1. Thiamine pyrophosphate (TTP)
    2. Flavin adenine dinucleotide (FAD)
      3. Nicotinamide adenine dinucleotide (NAD)
      4. Coenzyme A (CoA)
      5. Lipoate
184
Q

5 steps of PDHC?

A
  1. E1 grabs acetyl group (acetyl group transferred to TPP of E1, CO2 released)
  2. Acetyl passed from E1 to E2 (acetyl group esterified to lipoate of E2)
  3. Acetyl bound to acetyl-CoA (acetyl group transesterified to CoA and released as acetyl-CoA, BUT E2 is now not functional as lipoate is reduced)
  4. Reduced lipoate oxidised back to lipoate and hydrogens are transferred to FAD to form FADH2 by E3
  5. Electrons transferred from FADH2 attached to E3 to NAD+ to form NADH and H+
185
Q

Why can the acetyl group be added to lipoate?

A

Lipoate has an S-S bond that can be broken so acetyl group can bind

186
Q

Why can intermediates not diffuse away from PDHC?

A

They are covalently bound

187
Q
  • Why is lipoate flexible?
  • Why is this useful?
A
  • Has a lysine side chain
  • Can move to 3 different active sites
188
Q

Substrate channelling:
- What does it allow?
- What does it prevent?

A
  • Several steps to proceed at a rate not limited by the free concentration of substrate
  • Side reactions
189
Q
  • Acetyl-CoA structure?
  • 2 parts it has?
A
  • CH3 - C = O
    |
    S - CoA
  • 4-phosphopantetheine part and 3’-phosphoadenosine diphosphate part
190
Q

What part of acetyl-CoA is fully oxidised?

A

Acetyl group (CoA is reused)

191
Q

What do some intermediates in the citric acid cycle (CAC) act as?

A

Signalling molecules controlling the cell

192
Q

3 roles of the CAC?

A
  1. Final oxidation pathway for all fuel molecules
  2. Generates ATP and ‘high energy’ electron carriers that can produce ATP
  3. Produces intermediates that can feed biosynthetic pathways
193
Q

3 oxidation reactions in the CAC?

A
  1. CO2 release via decarboxylation
  2. C=C bond formation
  3. Removal of hydroxyl groups
194
Q

5 steps of CAC?

A
  1. Acetyl-CoA + oxaloacetate –> citrate
  2. Citrate rearragement to isocitrate
  3. Isocitrate –> 2-oxoglutarate
  4. 2-oxoglutarate –> succinyl-CoA
  5. 4 step rearrangement
195
Q
  1. Acetyl-CoA + oxaloacetate –> citrate:
    - Enzyme?
A
  • Citrate synthase
196
Q
  1. Citrate rearragement to isocitrate:
    - Why must it be rearranged?
    - How is it rearranged?
    - Enzyme?
A
  • Citrate is hard to oxidise as carbon that is partially oxidised has no H available to remove
  • OH on different carbon as H2O is removed and re added elsewhere
  • Aconitase
197
Q
  1. Isocitrate (6C) –> 2-oxoglutarate (5C)
    - What is used?
    - What is released?
    - Enzyme?
A
  • NAD+ (2H released from isocitrate to form NADH + H+)
  • CO2
  • Isocitrate dehydrogenase
198
Q
  1. 2-oxoglutarate (5C) –> succinyl-CoA (4C)
    - What is used?
    - What is released?
    - Enzyme?
A
  • CoASH
  • CO2 and NADH + H+
  • 2-oxoglutarate dehydrogenase complex
199
Q
  1. 4 step rearrangement (STEP 1):
    - What occurs?
    - What is used?
    - What is released?
    - Enzyme?
A
  • Succinyl-CoA to succinate
  • ADP + Pi
  • CoASH
  • Succinate thiokinase
200
Q
  1. 4 step rearrangement (STEP 2):
    - What occurs?
    - What is used?
    - Enzyme?
A
  • Succinate to fumarate
  • FAD
  • Succinate dehydrogenase
201
Q
  1. 4 step rearrangement (STEP 3):
    - What occurs?
    - What is added?
    - Enzyme?
A
  • Fumarate to malate
  • H2O
  • Fumerase
202
Q
  1. 4 step rearrangement (STEP 4):
    - What occurs?
    - What is used?
    - Enzyme?
A
  • Malate to oxaloacetate
  • NAD+ (2H lost from malate)
  • Malate dehydrogenase
203
Q

What does one turn of the CAC form?

A

3 NADH, 1 FADH2, 1 ATP, 2 CO2

204
Q

What is the main energy storage in fats?

A

Triglycerides

205
Q

Dehydrogenation of glycerol:
- Products?
- Side-products?
- Enzymes?

A
  • DHAP (glycolytic intermediate)
  • ATP and NADH + H+
  • Glycerol kinase and glycerol-3-phosphate dehydrogenase
206
Q

What process breaks down fatty acids?

A

Beta-oxidation (e.g. C16 fatty acid forms 8 acetyl-CoA)

207
Q
  • What is transamination?
  • What is reverse-transamination?
A
  • Amino acid oxidation to produce glycolytic intermediates (alanine to pyruvate, aspartic acid to oxaloacetate)
  • The reverse
208
Q

What does oxaloacetate act as?

A

The primary starting material for all carbohydrate forms through gluconeogenesis

209
Q
  • How much ATP does 1 glucose molecule generate in the CAC?
  • In glycolysis?
A
  • 25
  • 7
210
Q
  • Why must CAC be regulated?
  • When does most of the regulation occur?
A
  • More energy is needed in different circumstances
  • First half
211
Q

Regulation of PDHC:
- How is it regulated?
- What inhibits it?
- What activates it?
- What enzyme inactivates it?
- What enzyme activates it?

A
  • Allosterically by phosphorylation status
  • ATP, acetyl-CoA and NADH
  • AMP, CoA, NAD+
  • Kinase phosphorylates it (inhibited by substrates and Ca2+, activated by products)
  • Phosphatase dephosphorylates it (stimulated by insulin and Ca2+)
212
Q
  • What are the key sites for regulation of the CAC?
  • 3 examples?
A
  • Reactions with large free energy changes
    1. Citrate synthase
    2. Isocitrate dehydrogenase
    3. 2-oxoglutarate dehydrogenase
213
Q

What does NADH and NAD+ affect?

A

High concentration can limit dehydrogenase enzymes

214
Q

What limits citrate synthase?

A

Low concentration of citrate

215
Q

What allosterically activates and inhibits isocitrate dehydrogenase?

A

Activated by ADP
Inhibited by NADH and ATP

216
Q

What inhibits 2-oxoglutarate dehydrogenase?

A

Its products such as succinyl-CoA and NADH

217
Q

What are anaplerotic reactions?

A

Reactions to remove and top-up CAC intermediates

218
Q
  • What is the major anaplerotic reaction?
    -Enzyme?
  • What stimulates the enzyme?
A
  • Pyruvate to oxaloacetate using CO2 and releasing ATP
  • Pyruvate carboxylase
  • Acetyl-CoA (regulates if pyruvate becomes acetyl-CoA or is carboxylated)
219
Q

Pyruvate carboxylase:
- What is its cofactor?
- Why is it multifunctional?
- 2 steps?

A
  • Biotin (attached to lysine side chain of biotin carboxyl carrier)
  • Has 2 active sites
    1. Carboxylates biotin (CO2 attaches to NH on biotin)
    2. Transcarboxylates pyruvate to oxaloacetate
220
Q

Mechanism of biotin gaining CO2?

A

As it can move, it gains CO2 from one component of enzyme and delivers it to another

221
Q

Pyruvate carboxylase:
- 2 domains?
- What does it form?

A
  • Biotin carboxylation domain and carboxytransferase domain
  • Homotetramer
222
Q

2 anaplerotic reactions in CAC in animals?

A
  1. Pyruvate + HCO3- + ATP –> oxaloacetate + ADP + Pi
    Catalysed by pyruvate carboxylase
  2. Phosphoenolpyruvate + CO2 + GDP –> oxaloacetate + GTP
    Catalysed by PEP carboxykinase
223
Q

What can phosphoenolpyruvate (PEP) enter using PEP carboxykinase?

A

CAC

224
Q

What is the anaplerotic reaction in plants yeast and bacteria?

A

PEP + HCO3- –> oxaloacetate + Pi
Catalysed by PEP carboxykinase

225
Q

What is the anaplerotic reaction in eukaryotes and bacteria?

A

Pyruvate + HCO3- + NAD(P)H –> malate + NAD(P)+
Catalysed by malic enzyme

226
Q

What is the glyoxylate cycle?

A

Cycle where plants and bacteria convery acetyl-CoA to sugars - bacteria can grow on acetate and plant seeds can make acetyl-CoA

227
Q

Normal order in CAC?

A
  1. Acetyl-CoA + oxaloacetate
  2. Citrate
  3. Isocitrate
  4. 2-oxoglutarate (CO2 released)
  5. Succinyl-CoA (CO2 released)
  6. Succinate
  7. Fumarate
  8. Malate
  9. Oxaloacetate
228
Q

What alterations does the glyoxylate cycle make to the CAC?

A
  • Uses isocitrate lyase to convert isocitrate to succinate (reenters cycle) and glyoxylate (2C), the glyoxylate then in turned into malate using malate synthase (acetyl-CoA split and acetyl group added to glyoxylate)
  • Also makes 2-oxoglutarate and succinyl-CoA irrelevant
229
Q

How do plants regulate the balance of the glyoxylate cycle and the CAC?

A

Separate glyoxysome from mitochondria

230
Q

How bacteria regulate the balance of the glyoxylate cycle and the CAC:
- What are the only enzymes they have when growing on acetate?
- What can occur in response to substrate?
- Where are both enzymes?
- How is isocitrate dehydrogenase regulated in the CAC?

A
  • Isocitrate lyase and malate synthase
  • Turning on and off of genes
  • In the same compartment
  • Phosphorylated by kinase to inactivate and dephosphorylated by phosphatase to activate
231
Q

What does pyruvate carboxylase require for catalysis?

A

An enzyme-attached biotin mioety

232
Q

What are aldotetroses unable to do?

A

Form ring structures as they are not stable enough

233
Q

What remains constant in the steady-state assumption?

A

[ES] over time of measurement

234
Q

How does increasing the temperature affect enzymes?

A

Increases mobility of internal tertiary structure

235
Q
A

Biomed (8 decks)

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