lecture IV: enzyme kinetics

Question 1

Enzyme

Answer 1

Enzymes are proteins, which catalyze chemical reactions in water under mild conditions of temperature and pH by lowering the activation energy. They speed up the approach of an equilibrium between substrate and product.

Question 2

Do enzymes affect equilibrium?

Answer 2

No!
They speed up the reaction, but will not change equilibrium (as that is determined by the energy states between S and P)

Question 3

Enzymes: exception

Answer 3

Catalytic RNAs (ribozymes) are not proteins, but function similarly to other enzymes.

Question 4

What is ΔG?

Answer 4

The change in free energy.

→ΔG = P - S

Question 5

Activation energy

Answer 5

The minimum amount of energy that is required to activate atoms or molecules to a condition in which they can undergo chemical transformation or physical transport.

→S to transition state

Question 6

What effect do enzymes have on the rate of reaction?

Answer 6

They will increase the speed since they facilitate the transition state.

Question 7

Transition state (T)

Answer 7

A substrate needs to undergo a structural change before being converted to a product. At the transition state, it is equally likely that the molecule decays to either substrate or product.

→not a stable reactant intermediate
→transient structure before bonds break/form
→high E state

Question 8

The lower the transition state after adding an enzyme, the _____ the enzyme

Answer 8

faster

Question 9

How do enzymes facilitate the transition state?

Answer 9

1. Provide a reaction surface and good environment
   →optimally orient reactants
2. Position reactants to easily attain their transition state
3. Weaken bonds
4. May participate in the reaction
5. Form a strong interaction with T, rather than S or P

you need slight affinity for both substrate and product with the enzyme, but never as much as with the transition state and the enzyme

Question 10

Is the active site usually more hydrophobic or hydrophilic?

Answer 10

hydrophobic!!

Question 11

What would happen if an enzyme had a higher affinity for S than for T?

Answer 11

No catalysis.

→If the substrate had more affinity then more IMF between enzyme and substrate, rather than transition so it would just stay there and there would be no catalysis

Question 12

What would happen if an enzyme had a higher affinity for P than for T?

Answer 12

Product inhibition.

→If the product had more affinity then you would get product inhibition since products need to be able to diffuse out of the enzyme

Question 13

Metal stickase enzyme example

Answer 13

Fun! go read slide 14 :)

Question 14

Pyruvate & Lactate Dehydrogenase reaction

Answer 14

Pyruvate + NADH + H+ ⇄(LDH)⇄ lactate + NAD+

Question 15

Equilibrium reaction

Answer 15

E+S ⇄ (1) ⇄ ES ⇄ (2) ⇄ EP ⇄ (3) ⇄ E+P

→(1) first energy minimum
→(2) transition state
→(3) second energy minimum

Question 16

Protease

Answer 16

Cleaves a peptide bond by hydrolysis

Question 17

Serine protease

Answer 17

Makes use of active serine for catalysis

Question 18

Explain the catalytic triad of serine proteases.

Answer 18

Slides 18-19

Question 19

Which AAs does the catalytic triad of serine proteases consist of?

Answer 19

1. Aspartic acid
2. Histidine
3. Serine

Question 20

Alkaline phosphatase assay

Answer 20

A colorimetric assay.

NPP (colorless) → NP (yellow)

→the more colour (higher absorbance), the more product, the more the enzyme is working

Question 21

Initial velocity (v0)

Answer 21

The initial velocity is the initial slope of a graph of the concentration of reactants or products as a function of time.

• at the beginning of the reaction (not at equilibrium)
• units: M/min OR mol/(L*min)

Question 22

Michaelis-Menten plot

Answer 22

The reaction rate as function of substrate concentration.

Question 23

KM

Answer 23

Michaelis constant.

→units: M
→[S] at half-maximal reaction rate (Vmax)
→the substrate concentration at which 50% of active sites are occupied
→tells us about the efficiency of enzyme

Question 24

Vmax

Answer 24

Vmax is the reaction rate when the enzyme is fully saturated by substrate, indicating that all the binding sites are being constantly reoccupied.

Question 25

What does a high KM indicate?

Answer 25

-weak binding
-inefficient enzyme

Question 26

What does a low KM indicate?

Answer 26

-high affinity
-fast enzyme

Question 27

Michaelis-Menten formula

Answer 27

E+S ⇄ (k1f & k2r) ⇄ ES →k3→ E+P

Question 28

KM equation

Answer 28

KM = (k2 + k3) / k1

Question 29

Michaelis-Menten equation: v0

Answer 29

v0 = (Vmax * [S]) / (KM + [S])

Question 30

Lineweaver-Burk plot

Answer 30

Produces a linear relationship of enzyme kinetics by graphing the double-reciprocal of the MM plot.

→ 1/[S] vs. 1/v0
→ slope = KM/Vmax
→y-intercept = 1/Vmax
→x-intercept = -1/KM

Question 31

Examples: enzymes & their KMs

Answer 31

1. Hexokinase on ATP → very low (0.4 mM)
2. Chymotrypsin on glycyltyrosinylglycine → high (108 mM)

Question 32

Types of enzyme inhibition

Answer 32

1. Competitive inhibition
2. Non-competitive inhibition
3. Uncompetitive inhibition
4. Irreversible inhibition
5. Suicide inhibition

Question 33

Competitive inhibition

Answer 33

Reversible inhibitor through (non-covalent) intermolecular forces.

→acts as if less substrate would be present
→increasing the substrate concentration will compete with inhibitor, making [I] less effective

• same Vmax
• KM increases

Question 34

Competitive inhibition: Draw MM & LWB plots for different [I] as [S] increases

Answer 34

L4 review, slide 8

Question 35

Competitive inhibition: example

Answer 35

• statins
• COX inhibitors

Question 36

Uncompetitive inhibition

Answer 36

Takes place when an enzyme inhibitor binds only to the complex formed between the enzyme and the substrate (ES complex).

→increasing substrate concentration does not compete with inhibitor (may actually encourage inhibitor since complex formation occur faster)
→rare for them to be effective at high []

• Vmax decreases
• KM decreases

Question 37

For uncompetitive inhibition, when is the inhibitor most effective?

Answer 37

At high substrate concentrations, since it promotes the formation of the ES complex.

Question 38

Uncompetitive inhibition: Draw MM & LWB plots for different [I] as [S] increases

Answer 38

L4 review, slide 10

Question 39

Non-competitive inhibition

Answer 39

Inhibitor binds at a site different from the active site (allosteric site).

→binding to the enzyme can happen in unbound or bound to substrate
→increasing substrate concentration has no effect
→effect on catalysis, not on active site conformation

• Vmax decreases
• same KM

Question 40

Non-competitive inhibition: Draw MM & LWB plots for different [I] as [S] increases

Answer 40

L4 review, slide 12

Question 41

Irreversible inhibition

Answer 41

Special case where the substrate undergoes a covalent interaction with the substrate.

→cannot be a competitive inhibitor

Question 42

Irreversible inhibition: example

Answer 42

Penicillin.

Question 43

Suicide inhibition

Answer 43

Suicide inhibition is a form of irreversible inhibition in which the substrate in the first catalytic cycle is converted into a chemically reactive product which remains bound to the active site through covalent bonding. The enzyme is rendered permanently inactive.

Question 44

How can suicide inhibitors be used for research?

Answer 44

If suicide inhibitors can be fluorescently labeled, it is a smart way to see where in the cell an enzyme is actually active!

Question 45

Transition state inhibitors

Answer 45

Mimic the catalytic transition state with a higher affinity than substrate or product

→binds through non-covalent forces, but quasi irreversible binding

Question 46

Transition state inhibitor: example

Answer 46

HIV protease inhibitor.

Indinavir mimics the tetrahedral state, such that the bond that would normally be cleaved, does not.

Question 47

Which parameters are good measures of efficacy?

Answer 47

1. KI
2. IC50
3. EC50

Question 48

KI

Answer 48

Dissociation constant of an inhibitor.

→ KI = ([E]*[I]) / [EI]

Question 49

The _____ the KI, the more potent the inhibitor.

Answer 49

lower!

Question 50

IC50

Answer 50

Inhibitory concentration 50.

→Inhibitor concentration inhibiting 50% of enzyme
→ IC50 = KI + ([total E]/2)

Question 51

How can IC50 be experimentally measured?

Answer 51

Assay where you have purified enzyme in a vial and then you add inhibitor.

Question 52

EC50

Answer 52

Effective concentration 50.

Inhibitor concentration to reduce a cellular effect by 50%.

Question 53

How can EC50 be experimentally measured?

Answer 53

Need to conduct a cell assay, and not just with pure enzyme.