CELLS

Question 1

Give 3 examples of inherited diseases involving enzyme defects?

Answer 1

• Phenylketonuria: lack of liver enzyme phenylalanine hydroxylase = can’t convert phenylalanine to tyrosine = phenylalanine accumulates and produces toxic by products
• Glycogen Storage disease: can’t mobilise glucose from glycogen
• Tay Sachs Disease: enzyme defect in processing a membrane ganglioside = complex lipid = neuronal damage and death

Question 2

Name 3 drug types that target enzyme action?

Answer 2

• Antibiotics
• Anti-inflammatory agents
• Anti-cancer drugs

Question 3

List 5 key properties of enzymes?

Answer 3

• Increase rate of reaction by up to 10 billion fold
• Show specificity
• Unchanged at end
• Does not alter equilibrium
• Decreases activation energy (deltaG) of reaction to facilitate reaction

Question 4

What are two investigations used to providence evidence of active sites?

Answer 4

• X-ray crystallography

- Kinetic studies of enzyme activity

Question 5

How do enzymes reduce DeltaG to help catalysis?

Answer 5

• Brings molecules close together in active site
• Strains bonds in the substrate to break it up
• Stabilises charges in transition state
• Excludes water from active site which speeds up reaction
• Provides reaction pathway with lower energy
• Uses co-factors

Question 6

What information does the Vmax provide?

Answer 6

Vmax/[enzyme] = Kcat (what occurs when enzyme is fully working and all active sites are filled)
- Kcat also referred to as turnover number = max no. of substrate molecules handled per active site per second

Question 7

What information does Km provide?

Answer 7

A measure of the affinity of the substrate for the active site

Question 8

Describe competitive inhibition and its effects on Vmax & Km?

Answer 8

Inhibitor competes with substrate for the active site

Vmax doesn’t change as high substrate concentration can outcompete effects of inhibitor

Km has to increase as more substrate is needed to achieve half of Vmax

Question 9

Descrive non-competitive inhibition?

Answer 9

Inhibitor binds to different site to substrate’s active site

Vmax is reduced as inhibitor is altering catalytic activity of substrate

Km is unaltered as substrate still has same affinity for active site

Question 10

What are the 4 ways that enzymes are regulated in cells?

Answer 10

• Controlled by gene expression
• Compartmentation: target enzymes to specific organelles
• Allosteric regulation: changes conformation of active site and decreases enzyme activity
• Covalent modification of enzyme: changes enzyme shape and activity

Question 11

Name an example of allosteric regulation

Answer 11

End product inhibition; end product has a different shape to first substrate, so binds to allosteric site

Question 12

List the properties of allosteric enzymes?

Answer 12

• Multisubunit complexes
• Regulatory sites and catalytic sites on different subunits
• Regulation occurs via conformational changes
• Involved in feedback inhibition of metabolic pathways

Question 13

What is the role of the bacterial DNA gyrase enzyme?

Answer 13

• Uses ATP to catalyse the conversion of relaxed DNA to supercoiled DNA
• Found in all bacteria and essential for DNA replication

Question 14

Describe some inhibitors that work against DNA gyros?

Answer 14

• Novobiocin; antibiotic that competitively inhibits the binding of ATP to gyrase
• Fluoroquinolones: antibacterial drug that blocks the breakage reunion activity of DNA gyros = essential for supercoiling

Question 15

Name 4 types of enzymes in cells and their function?

Answer 15

Proteases - hydrolyse peptide bonds in proteins
Nucleases - break down nucleic acids; RNA/DNA
Polymerases - catalyse reactions between nucleotides to form polymers; RNA/DNA
Kinases - transfer phosphate from ATP to a substrate

Question 16

Give examples of different functions enzymes can carry out?

Answer 16

• Digestion
• Blood clotting
• Defence
• Movement
• Nerve conduction

Question 17

How would you define a “perfect” enzyme?

Answer 17

An enzyme where reaction rate is limited by diffusion of enzyme and substrate together (E+S)

Evolution has sped up the chemical reactions of the enzyme (ES to EP), making them no longer the rate limiting part of the enzyme

Question 18

Describe the 3 ways you can check if an enzyme reaction is limited by diffusion?

Answer 18

1. Monitor rate of reaction when combining enzyme with small amount of substrate
   - If diffusion is rate limiting, it should be decreasing when viscosity of solution is increased (e.g. adding glycerol)
2. Using theoretical calculations which takes into account size of enzyme and substrate
   (k3/Km)
   - k3: rate constant of ES to EP reaction
   - Km: substrate conc where rate is at half of Vmax
3. Determine the full free energy profile for reaction = tells us whether substrate binding is rate limited

Question 19

Name an example of a “perfect” enzyme?

Answer 19

Triosephosphate isomerase

Question 20

What are the different active sites that proteases can use?

Answer 20

serine
cysteine
aspartyl
metal iron

Question 21

How do serine proteases work?

Answer 21

Contain very active serine at active site = serine reacts with substrate to produce an acyl enzyme intermediate = hydrolysed by water

Hydrolyses peptide bonds but with sequence specificity - determined by the residue on the N terminal which is going to be cleaved

Question 22

Describe how Chymotrypsin, Trypsin and Elastase exhibit their substrate specificity?

Answer 22

Chymotrypsin - only cleaves peptide bonds where Phe, Trp or Tyr are the residues at the N-terminal (all hydrophobic)

Trypsin - only cleaves peptide bonds where Lys or Arg are the residues at the N-terminal (positive charged amino acids)

Elastase - only cleaves peptide bonds of very small residues