CW5-6: Enzymes

Question 1

What makes up the ‘Y’ shape of an antibody such as IGG?

Answer 1

• 2x F_ab domain (containing antigen-binding sites)
  
  • Contains a light chain and a heavy chain
• 1x F_c domain
  
  • Contains a heavy chain

Question 2

How can myosin be studied?

Answer 2

It can be cut into four strands.

• Trypsin can be used to cut the myosin into heavy meromyosin (HMM) and light meromyosin (LMM)
• Papain then cleaves off the head section, resultin in two parts

Question 3

Define ‘heptad’.

Answer 3

A repeated sequence of seven amino acids.

Question 4

How many amino acids form one full turn of an alpha helix?

Answer 4

3.6

Question 5

If a right-handed alpha-helical coiled-coil is a heptad in length, how many turns is it?

Answer 5

2

Question 6

How does carboxypeptidase hydrolyse other proteins?

What prevents it attacking itself?

Answer 6

It attacks them at their C-terminus.

Its C-terminus is hidden within the folds of the protein.

Question 7

What is an enzyme?

Answer 7

A protein which catalyses biological reactions

Question 8

What are the properties of an enzyme?

Answer 8

• Highly specific (only some substrates can fit into the active site)
• Unchanged after reaction (regenerated at the end)
• Can accelerate reactions up to 10⁶-fold
• Cannot alter reaction equilibria (will end up at teh same point in both the presence and absence of catalyst, energetically speaking)

Question 9

What’s the general equation for the conversion of substrate to product?

Answer 9

S ⇌ P

Question 10

What are the two factors that determine the appearance of a product?

Answer 10

• Thermodynamic factors (energy changes)
• Kinetic factors (rate changes)

Question 11

What must the value of ΔG be for a reaction to occur?

Answer 11

Negative

Question 12

What effect does a catalyst have on activation energy (E_A)?

Answer 12

A catalyst lowers the E_A required, so the reaction occurs more easily.

Question 13

What is the shape of the graph of substrate concentration vs. rate?

Answer 13

Rectangular hyperbola

Question 14

How is K_M important in relation to glucose?

Answer 14

Hexokinase has a K_M of 0.1 mM, so when the concentration of glucose is low, more glucose is converted to glucose-6-phosphate, resulting in energy release. A build-up of G6P results in inhibition of hexokinase.

Glucokinase (found only in the liver) has a K_M of 5 mM, so when the level of glucose reaches this level, G6P is formed and stored as glycogen in the liver.

Question 15

What is the ratio K_cat:K_M a good measure of?

Answer 15

Enzyme efficiency

An efficient enzyme has a high ratio (either high K_cat or low K_M)

Question 16

What is the active site of an enzyme?

Answer 16

A region where reactions occur. In general, active sites form only a small section of an enzyme. They create a microenvironment, e.g. they often exclude water.

Question 17

What are the two theories of enzyme-substrate complex formation?

Answer 17

1. Lock and key (Emil Fischer, 1890)
2. Induced fit (Dan Koshland, 1958)

Question 18

What are the issues with the lock and key theory of enzyme-substrate complex formation?

Answer 18

• There is no reason to suggest products want to be formed because they fit together snugly so there is no explanation for the equation:

ES → E + P₁ + P₂

• Why would the binding of a non-competitive inhibitor away from the active site prevent the enzyme doing its job?

Question 19

What did Dan Koshland propose was a better model for enzyme-substrate complexes?

Answer 19

The induced fit model, where the intermediate is presented as a transition state conformation. This is under tension so it is more likley to form products.

This theory fits better with the data, and non-competitive inhibitors can now be rationalised and their activity explained.

Question 20

What are the two main types of enzyme inhibitor? Describe both.

Give the two subgroups of one type of inhibitor.

Answer 20

1. Irreversible – bind very tightly and generally form covalent bonds
2. Reversible – non-covalent
   
   1. Competitive – binds at the active site
   2. Non-competitive – binds at another site on the enzyme

Question 21

How do nerve gases such as sarin work?

Answer 21

They are irreversible enzyme inhibitors. Sarin is an acetylcholinesterase inhibitor, so nervous signals do not stop and muscle contraction is permanent.

Question 22

What effect do competitive inhibitors have on V_max and K_M?

Answer 22

V_{<strong>max</strong>} unchanged because a high enough substrate concentration can overcome the inhibitor

K_M increases because a higher substrate concentration is required to reach 0.5V_max

Question 23

What effect do non-competitive inhibitors have on V_max and K_M?

Answer 23

V_max decreases because adding more substrate won’t overcome the effect of the inhibitor becuase the inhibitor binds to another part of the enzyme

K_M will remain the same (see M-M graph)

Question 24

Describe ribonucelase.

Answer 24

• 124 amino acids (13.7 kDa)
• Hydrolyses phosphodieaster bonds in RNA
• Releases free 3’-phosphate and 5’hydroxyl termini
• Bell-shaped pH curve with optimum around pH 7
  
  • Tighter curve than proteins in general
• Catalysis by a pair of histidine residues

Question 25

Describe the process of the alkali-catalysed hydrolysis of RNA.

Answer 25

• The deprotonated 2’ OH of the ribose acts as a nucleophile and attacks the adjacent phosphorus in the phosphodiester bond of the sugar-phosphate backbone of the RNA.
• There is a transition state, where the phosphorus is bonded to five oxygen atoms.
• The phosphorus then detaches from the oxygen connecting it to the adjacent sugar, resulting in ester cleavage of the RNA backbone.
• This produces a 2’,3’-cyclic phosphate that can then yield either a 2’- or a 3’-nucleotide when hydrolysed.

Question 26

Which amino acids are involved in the mechanism of ribonuclease? Which is protonated and which is unprotonated?

Answer 26

His 12 – unprotonated so accepts proton

His 119 – protonated so donates proton

Question 27

What is the evidence for the mechanism of ribonucleases?

Answer 27

1. pH-dependence of reaction (tight bell curve)
2. Use of small molecule inhibitor, e.g. iodoacetate
3. Use of a non-hydrolysable substrate analog, e.g. altered ribonuclease
4. Site-directed mutagenesis (null reaction uses alanine instead because it’s a typical non-reactive amino acid)

Question 28

Why is the bell curve for the pH dependence of ribonuclease activity so tight?

Answer 28

The pK of histidine is around 6.5, so it is readily protonated/unprotonated

Small pH changes result in both histidine residues being protonated or unprotonated so there is no reaction

Question 29

How does iodoacetate carboxymethylate histidine?

Answer 29

It donates an acetate (CH₂COO^–) to one of the histidines

Question 30

What are substrate analogs? Give an example.

Answer 30

Chemical compounds with a chemical structure that resembles the substrate molecule in an enzyme-catalyzed chemical reaction. They can act as competitive inhibitors of an enzymatic reaction.

e.g. phosphonate is a substrate analog for phosphate ester. The enzyme thinks it’s the right substrate but cannot let it go because it cannot be processed by the enzyme due to the CH₂ group (where the O would normally be)