Molecular Biology and BioChem 1

Question 1

What is the difference between tertiary and secondary structure?

Answer 1

Tertiary structure is the coming together of all the elements of secondary structure to form a complete native folded protein
Secondary structure is α helix and β sheet, the 3-D structure of local segments

Question 2

What forces drive the formation of tertiary structure?

Answer 2

The same forces that drive secondary structure formation
For example:
Hydrogen bonding
Salt bridges
Hydrophobic effect
Disulphide bridges

Question 3

What is the advantage of haemoglobin being able to bind 4 haem groups?

Answer 3

It has a very fine control over the binding of oxygen. The binding of oxygen onto the first haem group affects the binding of oxygen onto the other haem groups.

Quaternary structure can be crucial to the function of proteins

Question 4

What are the main methods of determining protein structure?

Answer 4

X-ray Crystallography- Reveals atomic structure- not all proteins can be crystallised.
NMR Spectroscopy- Similar level of detail as X-ray Crystallography but you don’t need crystals- can only solve small proteins.
Cryo-electron microscopy- You don’t need crystals and can be used for large structures- not that good at small proteins.

Question 5

For membrane-bound proteins where does the hydrophobic section of the protein sit?

Answer 5

The hydrophobic section of the protein will sit within the membrane.

Question 6

What are polar residues within the transmembrane α helices indicative of?

Answer 6

A channel

Question 7

What are the key characteristics of membrane-spanning α helices?

Answer 7

Very hydrophobic
About 20 amino acids long
Not many polar residues

Question 8

What is the difference between integral membrane proteins and peripheral membrane proteins?

Answer 8

Integral membrane proteins span the membrane, and so tend to be rich in α helices.
Peripheral membrane proteins associate with head groups of the membrane or integral membrane proteins or associated with fatty acid groups in the membrane.

Question 9

Describe how proteins fold

Answer 9

Progressive stabilisations of small regions. The most stable secondary structures fold first and other structures fold against. Fold in an energetically favourable way to maximise non-covalent bond formation.

Question 10

Describe the findings of the Anfinsen Experiment.

Answer 10

All the information about the tertiary structure is found in the primary structure
When you remove the denaturing agents the protein can refold.

Question 11

Discuss what cofactors and what prosthetic groups are and what the difference is between them

Answer 11

Cofactors are small organic compounds or metals that help to enhance the diversity of the functional groups in proteins
Prosthetic groups are permanently associated cofactors

Question 12

Give an example of a cofactor and describe what it does

Answer 12

Haem- in haemoglobin - metal (iron)- helps with the carrying of oxygen
NAD+- accepts OH- groups and then donates it later- acts as a transient carrier of important functional group

Question 13

What do Zinc fingers bind to? Where do they insert?

Answer 13

They bind to DNA - they insert into the major groove

Question 14

What are the different types of post-transcriptional modification and how to they change to properties of the protein?

Answer 14

Glycosylation- addition of carbohydrate group- makes proteins more hydrophilic and more soluble
Hydroxylation- addition of a OH group- increases H-bonding potential- makes the protein more rigid, can form stronger structure e.g. collagen
Phosphorylation-addition of phosphate group from ATP- adds to negative charge- changes charge distribution- can change the structure of a protein
Acetylation- addition of acetyl group- neutralises positive charge from lysine- causes DNA to be less densely packed around histones and be more accessible

Question 15

What is specific activity in terms of protein purification?

Answer 15

Number of enzyme units per milligram of total protein.

Question 16

What are some key factors to consider when planning an experiment and deciding on how to purify a protein?`

Answer 16

Yield vs purity (specific activity)
The source of the protein
Solubility, size, charge, surface hydrophobicity, binding for a ligand (of target protein)

Question 17

Describe the stages of liquid chromatography

Answer 17

Pass protein mixture through solid matrix of beads in a column. Choose beads depending on the property you want to exploit (charge/ size). Use UV light to detect protein peaks as fractions pass through the column
Proteins with a higher affinity for the matrix- bind with higher affinity- elute later
Ion-exchange chromatography- separate by charge
Size-exclusion chromatography- separate by size

Question 18

What is activation energy?

Answer 18

Activation Energy is the minimum amount of energy that is required to activate atoms or molecules to a condition in which they can undergo a chemical transformation.
In transition-state theory, the energy required to reach transition state.

Question 19

What is reaction rate?

Answer 19

Reaction rate is the rate at which reactants are converted to products.
Reaction rate can be described as the rate constant * concentration of reactants.
If X -> Y,
Rate = k[X]
(Square brackets = Concentration)

Question 20

What is the transition state?

Answer 20

In any chemical reaction, the reactants go through a transition state.
The Transition state the highest energy configuration of molecules throughout the process of the reaction.

Question 21

What are the best ways to speed up a reaction?

Answer 21

By changing heat and pressure.

This isn’t really possible under physiological conditions…
Instead, we use enzymes. Enzymes lower the activation energy under physiological conditions, by stabilising the transition state.

Question 22

What is Vmax defined as?

Answer 22

The maximum rate that a given concentration of an enzyme can achieve

Question 23

What is Km defined as?

Answer 23

Km is the Michaelis constant – substrate concentration at Vmax/2

Question 24

What are enzymes? How do they affect the rate of a reaction? How do they alter activation energy?

Answer 24

Enzymes are biological catalysts, they reduce the rate of reaction but are unchanged themselves. They reduce the activation energy by lowering the energy of the transition state.

Question 25

What is the Michaelis- Menten equation?

Answer 25

V = Vmax X [S] / Km + [S]

Question 26

What are the 3 assumptions made for the MM equation?

Answer 26

1. ) Progress from the activated enzyme-substrate complex to the formation of products is irreversible
2. ) Substrate is available in very large amounts compared to enzyme
3. ) The enzyme-substrate complex is at steady state (unchanging over time)

Question 27

Briefly explain the meaning of dynamic equilibrium and “steady state approximation”.

Answer 27

Dynamic equilibrium: The rate of the forwards and backwards reactions are equal- hence the relative concentrations of reactants and products are unchanging.
Steady state approximation: The concentration of the ES is low and constant due to a.) the slow complex formation and b.) the rapid consumption (once formed it is converted into products rapidly)

Question 28

True or false, “Km can be used as a measure of binding affinity?

Answer 28

True

Question 29

What does a large Km value suggest?

Answer 29

Large Km = weak binding (low affinity) and small Km = strong binding (high affinity)

Large Km = more substrate needed to achieve ½ Vmax = enzyme and substrate have low binding affinity for each other. Graph will look less steep.

Question 30

What is meant by the saturation of the enzyme? Where does it occur?

Answer 30

Occurs at Vmax. More substrates than active sites of enzyme.

“Maximum theoretical rate”

Question 31

What is Kcat? What are its units and how is it used?

Answer 31

Kcat: number of moles of product generated per mole of enzyme per second = rate of turnover = Vmax / E tot
Enzyme efficiency = Kcat / Km

Question 32

Why do you need catalytic efficiency to compare? Why not use simply Km?

Answer 32

Km only measures how much substrate is required to get enzyme activity. Varies widely. Different qualities required in different enzymes: substrate affinity, turnover rate, substrate specificity.
Enzyme efficiency = Kcat / Km. increase enzyme efficiency by increasing Kcat or reducing Km

Question 33

Describe the two models of enzyme binding.

What are the main differences?

Answer 33

Lock and key binding or induced fit.
In induced fit, the binding of a substrate induces a conformational change in the active site- the strain causes the substrate into a conformation similar to the TS.

Question 34

What is the benefit of transition state stabilisation?

Answer 34

The transition state is the highest energy state of the reaction and so the most unstable. Stabilising it will reduce the activation energy, so allow the reaction to occur at an increased rate. Note: Vmax is larger where TS energy is lower- the reaction goes faster with a stabilised TS.

Question 35

What are the two forms of Enzyme inhibition?

Answer 35

Competitive inhibition

Non-Competitive inhibition

Question 36

How do competitive and non-competitive inhibition differ?

Answer 36

Competitive inhibition is regulation by substrate analogues.

Non-competitive inhibition is regulation by binding at a distinct site called an allosteric site.

Question 37

What are the catalytic sites of ATCase?

Answer 37

The enzyme is made of catalytic trimer (catalytic) and regulatory dimer (binds inhibitor)
The Quaternary structure reorganises when there’s no ligand bound (no active site). Catalytic site lies between C subunits.

Question 38

What inhibits ATCase and how? What is the biological reason for this inhibition?

Answer 38

Regulated by CTP - end product in pathway to ensure correct amount of CTP is made in cell

T-state = poor substrate binding/activity, stabilised by inhibitor binding, prevents transition to R-state
R-state = highly active, binds substrate over inhibitor
R and T exist in equilibrium, CTP shifts equilibrium towards less active T-state, and ATP shifts the equilibrium towards the more active R state (this is in order to maintain the ratio between purines and pyrimidines).
Substrate binding at one active site causes other attached enzymes to bind substrate too.

Question 39

Describe what is meant by allosteric inhibition

Answer 39

Binding of an inhibitor at one site on a protein/enzyme which brings about an effect at a distance is allosteric inhibition

Question 40

Which ion is needed for DNA Polymerase activity?

Answer 40

Mg 2+

Question 41

During DNA replication, where are new nucleotides added?

Answer 41

3’ -OH of the previous nucleotide’s sugar group

Question 42

Which of these histone proteins does NOT form part of the octamer?

• H1
• H2A
• H3
• H4

Answer 42

H1 - It’s a linker protein

Question 43

Which of these protein structure determination methods can resolve larger
proteins, but not smaller ones?
-X-Ray Crystallography
-NMR
-Cryo-Electron microscopy

Answer 43

Cryo-Electron microscopy

Question 44

Which form of ATCase favours substrate binding?

• T Form
• S Form
• Q Form
• R Form

Answer 44

R Form

Question 45

Difference between Nucleoside and Nucleotide?

Answer 45

Nucleoside = base + sugar
Nucleotide = base + sugar + phosphate

Question 46

Which base pairing is the most stable?

Answer 46

C-G

This is because the pairing has 3 hydrogen bonds

Question 47

What forces are involved in base stacking?

Answer 47

Hydrophobic interactions

Base stacking reduces exposure of the hydrophobic bases to the aqueous environment

Question 48

Why is DNA better as an information storage substrate than RNA?

Answer 48

The O-H bond in the ribose of RNA makes the molecule more reactive, compared to DNA. RNA is unstable under alkaline conditions, and it is more susceptible to enzyme attack. RNA is degraded quickly.

Question 49

What are the forces contributing to the stability of DNA?

Answer 49

The stability of the DNA double helix depends on a fine balance of interactions including hydrogen bonds between bases, hydrogen bonds between bases and surrounding water molecules, and base-stacking interactions between adjacent bases.

Question 50

How does the proofreading subunit of DNA polymerase detect errors in DNA
replication?

Answer 50

By reading the minor groove of DNA and checking for structural deformities in double helix caused by DNA damage (e.g. wrongly incorporated bases)

Question 51

What is a nucleosome? Describe the structure of a nucleosome

Answer 51

The basic unit of DNA packaging - consists of octameric histone core around which DNA is wound (~146 bases wrapped around core) with additional linker DNA between nucleosomes.