Enzyme Kinetics Flashcards
What is the michaelis complex?
This is the enzyme substrate complex
E + S -> ES (reversible) -> E + P
What is the turnover number (kcat)
This is the number of substrate molecules that can be converted to product by 1 enzyme molecule in 1 second
What graph is draw to show the rate of reaction?
Velocity vs [substrate]
Velocity is the rate of reaction at a defined substrate concentration
What is the order at low substrate concentration?
At low substrate concentration,
v is proportional to [S] - first order kinetics
What is the order of the reaction at high substrate concentrations
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
What is vmax?
This is the maximum rate of reaction
What is Km?
This is the dissociation constant
This is how well the enzyme binds to the substrate- affinity
What is km in terms of vmax?
Km is vmax/2
What does a low value of km mean?
This means the enzyme has a high affinity for the substrate as it takes less substrate to reach vmax/2
What does a high value of km mean?
This means the enzyme has a low affinity for the substrate as it requires more substrate to reach vmax/2
What is the equation for kcat?
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
What is kcat in terms of rate constants
E+S -> (k1,k-1) ES-> E+P (k2)
If the enzyme is saturated so solely ES is present, kcat= k2
What is the michaelis & menten equation?
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
What are the michaelis & menten assumptions?
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
What is the specificity constant?
Kcat/ km
This is a measure of catalytic efficiency
When is the specificity constant used?
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)
What is the limit for the specificity constant kcat/km?
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
What is catalytic perfection?
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
What is an enzyme inhibitor?
Compounds that reduce the rate of an enzyme catalysed reaction (drug molecules)
- they reduce the rate an enzyme can catalyst a reaction
What are the types of enzyme inhibitors?
1) irreversible
2) reversible
- competitive
- non competitive
What are the characteristics of irreversible inhibitors?
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
What are prostaglandins?
Prostaglandins are chemicals involved in pain
How does aspirin work?
Aspirin blocks the action of the cyclooxygenase involved in prostaglandin synthesis by transferring an acetyl group to serine 350
Describe the action of aspirin
Serine 350 attacks the penicillin
Serine residue has been deactivated (in enzyme active site)
See mechanism
Describe the action of penicillin?
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
What is Penicillin resistance?
Resistance is caused by ring opening beta lactamases
How do you overcome penicillin resistance?
Overcome resistance by administering beta lactamases inhibitor- clavilanic acid
How can you exploit a nucleophillic serine?
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
What is an acetyl choline esterase inhibitor?
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
How are ACE inhibited by organophosphorous reagents
Malathion - pesticide- COCH3 leaving group
OH on serine attacks- residue is reduced
Inactivates the enzyme and inhibits acetylcholine
See mechanism
Describe the action of sarin?
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
What are the characteristics of competitive inhibitors?
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
What affect does a competitive inhibitor have to v vs [S] graph?
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
What are the characteristics of a non competitive inhibitor?
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
What affect does a non competitive inhibitor have to v vs [S] graph?
Km remains unchanged as the enzyme will still bind to substrate and still have the same affinity
Vmax decreases- as enzymes become inactive
