25. Enzymes II Flashcards
Describe Michaelis constant? (2pts)
- The substrate concentration at which half the active sites are occupied and therefore will see half the maximum rate. Measure of the affinity of the substrate for the enzymes active site
V= Vmax (S)/ (S) + Km
Describe perfection in enzyme catalysis? (4pts)
- Evolution acts to maximise catalytic activity.
- Perfect enzymes have reactions limited by diffusion
- Evolution can improve the chemistry of the reaction. Evolution cannot work on the physical nature of the difussion step so cannot affect the rate of which the enzyme and substrate find themselves in solution. The overall reaction rate is determined by the slowest step.
- The limiting step will eventually be the diffusion step.
How to check if the reaction rate is affected by viscosity?
- Add glycerol to the reaction mixture
- This makes the aqueous solution more viscous and slows down the rate at which E and S diffuse together to form ES.
- A change in viscosity slows down the enzyme reaction rate. If this happens the enzyme is diffusion limited and it is catalytically perfect.
How can we check if enzymes are diffusion limited and therefore perfect? (2pts)
- Turnover number
2. Km value
Describe Trisphophate isomerase?
A perfect enzyme as it shows that an enzyme is a diffusion limited system.
Describe Serine Proteases? (4pts)
- Serine proteases= Enzymes which hydrolyse peptide bonds in other proteins
- Serine proteases have a very reactive serine which attacks the peptide bond to form an acyl-enzyme.
- Serine proteases hydrolyse the protein peptide bonds with sequence specificity.
- Serine proteases do not hydrolyse every peptide bond.
Describe Chrymotrypsin
Chymotrpsin likes to cleave when residue is hydrophobic. It has a hydrophobic pocket which accommodates the residues.
Describe Trypsin
Trypsin likes to cleave positively charged residues. This is because it is negatively charged so forms electrostatic attractions.
Describe Elastase?
Elastase likes to favour a small residue. The binding pocket is narrow.
Describe the 2 step hydrolytic pathway for serine proteases? (2pts)
- Acylation
- enzyme uses serine-OH to attack the carbonyl group of the peptide bond
- this forms an ester bond and an Acyl enzyme intermediate - Hydrolysis of the acyl enzyme intermediate
- the peptide bond is hydrolysed by a serine protease.
Describe the charge relay system? (4pts)
- A charge-relay system activates the catalytic serine by proton withdrawal.
- it shifts protons away from the oxygen of serine
- The oxygen is very electronegative and is able to carry out nucleophilic attack on the peptide bond.
- The charge relay system can pull protons off oxygen.
Describe mitochondrial ATP synthase? (5pts)
- ATP synthesis in mitochondria occurs by a proton-driven rotary ATP synthase (stryer)
- Protons are pumped across this membrane as electrons flow through the respiratory chain.
- The outer membrane is permeable whilst the inner membrane is impermeable.
- Respiration forms a proton gradient across the inner mitochondrial membrane.
- There is a high hydrogen ion concentration on the outside and low hydrogen ion concentration on the inside.
Describe Rotary ATP synthase? (2pts)
- Rotary ATP synthase has 3 active sites activated by a rotating spindle
- Protons drive the rotation of the system which results in the production of ATP. This was first proposed by Paul Boya.
Describe ATP production by oxidative phosphorylation?
- The ADP and phosphate are bound at an active site.
- The rota then rotates
- This provides energy to release ATP that is previously made in another active site.
- This squeezes together ADP and phosphate to make ATP at the other active site.
How can rotation of ATP synthase be visualised? (3pts)
- Actin filament is attached
- When ATP is added the actin filaments rotate.
- This shows that this is an enzyme that rotates in the opposite direction when it synthesises ATP.
