Enzyme Kinetics

Question 1

What is the michaelis complex?

Answer 1

This is the enzyme substrate complex

E + S -> ES (reversible) -> E + P

Question 2

What is the turnover number (kcat)

Answer 2

This is the number of substrate molecules that can be converted to product by 1 enzyme molecule in 1 second

Question 3

What graph is draw to show the rate of reaction?

Answer 3

Velocity vs [substrate]

Velocity is the rate of reaction at a defined substrate concentration

Question 4

What is the order at low substrate concentration?

Answer 4

At low substrate concentration,

v is proportional to [S] - first order kinetics

Question 5

What is the order of the reaction at high substrate concentrations

Answer 5

This is because at high [S] all of the enzyme active sites are filled which means
This means that no more substrate can increase the rate and so the reaction is zero order with respect to [S]
- vmax

Question 6

What is vmax?

Answer 6

This is the maximum rate of reaction

Question 7

What is Km?

Answer 7

This is the dissociation constant

This is how well the enzyme binds to the substrate- affinity

Question 8

What is km in terms of vmax?

Answer 8

Km is vmax/2

Question 9

What does a low value of km mean?

Answer 9

This means the enzyme has a high affinity for the substrate as it takes less substrate to reach vmax/2

Question 10

What does a high value of km mean?

Answer 10

This means the enzyme has a low affinity for the substrate as it requires more substrate to reach vmax/2

Question 11

What is the equation for kcat?

Answer 11

Kcat= Vmax / [E]total

[E] is the concentration of the enzyme
Kcat is the number of reaction events (turnover) catalysed by each active site per unit time

Question 12

What is kcat in terms of rate constants

Answer 12

E+S -> (k1,k-1) ES-> E+P (k2)

If the enzyme is saturated so solely ES is present, kcat= k2

Question 13

What is the michaelis & menten equation?

Answer 13

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

V= velocity at specific substrate concentration
Vmax= maximum attainable velocity by an enzyme under given conditions
[S]= substrate concentration 
Km= Michaelas constant - affinity between enzyme and substrate

Question 14

What are the michaelis & menten assumptions?

Answer 14

1) the ES -> E+P(k2) is irreversible: initial rate conditions
2) assume that [ES] is constant (intermediate)- steady state conditions- assume that [ES] is formed as quickly as it is consumed so the fate of formation equals the rate of consumption

Question 15

What is the specificity constant?

Answer 15

Kcat/ km

This is a measure of catalytic efficiency

Question 16

When is the specificity constant used?

Answer 16

When the substrate concentration is low, very little ES is formed and then kcat isn’t a useful measure of catalysis
Kcat/km is the apparent second order rate constant which reflects this (because not the rate reflects how often substrate and enzyme encounter one another)

Question 17

What is the limit for the specificity constant kcat/km?

Answer 17

The ultimate limit for kcat:Km is set by k1, the rate of formation of ES
This cannot be faster than diffusion as enzymes and substrates have to find each other in solution

Question 18

What is catalytic perfection?

Answer 18

Enzymes with specificity constants equal to the rate of diffusion have achieved catalytic perfection
They perform catalysis every time a collision occurs between the substrate and enzyme
- this is limited by diffusion- how quickly they can find eAchother

Question 19

What is an enzyme inhibitor?

Answer 19

Compounds that reduce the rate of an enzyme catalysed reaction (drug molecules)
- they reduce the rate an enzyme can catalyst a reaction

Question 20

What are the types of enzyme inhibitors?

Answer 20

1) irreversible
2) reversible
- competitive
- non competitive

Question 21

What are the characteristics of irreversible inhibitors?

Answer 21

1) bind irreversible to an enzyme via a covalent bond (serine in active site)
2) bond to nucleophillic side chain
3) binding permanently inactivated the enzyme
4) prevents substrate binding because it usually binds to active site and so not substrates can access it

Question 22

What are prostaglandins?

Answer 22

Prostaglandins are chemicals involved in pain

Question 23

How does aspirin work?

Answer 23

Aspirin blocks the action of the cyclooxygenase involved in prostaglandin synthesis by transferring an acetyl group to serine 350

Question 24

Describe the action of aspirin

Answer 24

Serine 350 attacks the penicillin
Serine residue has been deactivated (in enzyme active site)
See mechanism

Question 25

Describe the action of penicillin?

Answer 25

Penicillin binds in the active site
The strain of the 4 membered lactam ring makes is very reactive
The serine is the side chain residue in activity site
It inhibits a transpeptidase involved in cell wall biosynthesis
See mechanism

Question 26

What is Penicillin resistance?

Answer 26

Resistance is caused by ring opening beta lactamases

Question 27

How do you overcome penicillin resistance?

Answer 27

Overcome resistance by administering beta lactamases inhibitor- clavilanic acid

Question 28

How can you exploit a nucleophillic serine?

Answer 28

Reactive serine (in enzyme active site) may be exploited for enzyme inhibitors 
Design inhibitors that mimic TS as these bond well to enzyme 
See mechanism

Question 29

What is an acetyl choline esterase inhibitor?

Answer 29

Acetyl choline is a neutrotransmitter that can communicate between chemical synapses
The ester is hydrolysed- switches off neurotransmission
Inhibition of of acetylcholine esterase stimulate last uncontrollable nerve cell action

Question 30

How are ACE inhibited by organophosphorous reagents

Answer 30

Malathion - pesticide- COCH3 leaving group
OH on serine attacks- residue is reduced
Inactivates the enzyme and inhibits acetylcholine
See mechanism

Question 31

Describe the action of sarin?

Answer 31

F- is the leaving group (much better)
Serine attacks and is reduced
Enzyme is inactivated- neutrotransimitters cannot switch off
- inhibitor binds to serine in active site
- chemical weapon
- see mechanism

Question 32

What are the characteristics of competitive inhibitors?

Answer 32

They bind to active site (similar structure to substrate or TS)

1) compete with substrate for access to active site
2) when bound they prevent binding of substrate
3) can be overcome by increasing [S] until it outcompetes the inhibitor

Question 33

What affect does a competitive inhibitor have to v vs [S] graph?

Answer 33

Add inhibitor
1) km increases (lower affinity of enzyme to substrate- takes longer to get to vmax/2
2) no change to vmax but takes more [S] to get to vmax
Add more substrate to overcome

Question 34

What are the characteristics of a non competitive inhibitor?

Answer 34

1) binds at site other than the active site- switches off catalysis
2) bond before or after the substrate binds
3) doesn’t prevent substrate from binding
4) prevents catalytic act from taking place
5) cannot be overcome by increasing [S]
6) only removed by repeated dialysis, cannot put compete

Question 35

What affect does a non competitive inhibitor have to v vs [S] graph?

Answer 35

Km remains unchanged as the enzyme will still bind to substrate and still have the same affinity
Vmax decreases- as enzymes become inactive