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Cell Phys > Lec 4: Enzymes (Pt. 2) > Flashcards

Lec 4: Enzymes (Pt. 2) Flashcards

1
Q

(Measuring Km & Vmax (Hexokinase))
Can’t easily obtain…
Can more easily obtain…

A

…the Km and Vmax when plotted as a hyperbolic function

…the Km and Vmax with the Lineweaver-Burke Plot

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2
Q

Enzyme Inhibitors =

A

Any chemical agent that decreases the activity of the enzyme by binding to (or interacting with) that enzyme

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3
Q

Why have enzyme inhibitors? (3)

A
  1. ) To control [S] or [P]
  2. ) Increase [S] unreacted
  3. ) Decrease [P] formed
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4
Q

Many ________ ______ can act as…

A

chemical agents

…inhibitors (even ATP, which inhibits PFK)

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5
Q

Each enzyme has

A

specific inhibitors (specificity)

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6
Q

(Alcohols) partake in…

and can…

A

…hydrogen bonding

…dehydrate tissue

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7
Q

Reducing agents can directly…

therefore…

A

…break disulfide bridges (the only covalent bond holding protein together)
…3D conf of protein is disrupted & t.f. nonfunctional protein

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8
Q

Inhibitors: 2 Main Categories

A
  1. ) Irreversible Inhibitors

2. ) Reversible Inhibitors

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9
Q

(Irreversible Inhibitors)

  1. ) cause…
  2. ) generally, they are…
  3. ) generally, they form…
  4. ) may act…
  5. ) consequently, they may not…
  6. ) The basic structure of the enzyme is…
  7. ) therefore, …
A
  1. ) …permanent loss of enzymatic activity.
  2. ) …toxic to cells.
  3. ) …strong bonds with an enzyme (may be covalent bonds).
  4. ) …at, near, or remote from the active site.
  5. ) …be displaced by the addition of excess substrate.
  6. ) …modified such that it ceases to work.
  7. ) …the enzymes won’t undergo the necessary conformational changes to catalyze its reaction.
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10
Q

Irreversible Inhibitors: (3)

& examples:

A
  1. ) Heavy metal ions
    ex: Ag+, Hg2+, Pb2+ have strong affinities for -SH groups.
  2. ) Organophosphates
    ex: nerve gas poisons & various insecticides, Diisopropylfluorophosphate (DFP), malathion, parathion, and chlorpyrifos
  3. ) Penicillin (antibiotic)
    ex: penicillin
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11
Q

(Irreversible Inhibitors)
Organophosphates inhibit…
by…

A

…the active site of acetylcholinesterase

…reacting with the hydroxyl group of serine permanently modifying it

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12
Q

(Irreversible Inhibitors)
Penicillin binds to…
forming…
and blocks…

A

…the bacterial enzyme (DD-transpeptidase)
…a highly stable penicilloyl-enzyme intermediate
…cell wall synthesis

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13
Q

Acetylcholine (Ach) is a…
located…
specifically, Ach functions as an…

A

…neurotransmitter
…in the central and autonomic nervous systems
…excitatory (stimulatory) neurotransmitter at the NMJ

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14
Q

Acetylcholinesterase (AchE) is…

and prevents…

A

…the enzyme that breaks down Ach, cleaving it into acetate and choline
…continued stimulation of muscle allowing it to relax.

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15
Q

(DFP inhibition of Acetylcholinesterase) Steps: (3)

A
  1. ) DFP enters the active site
  2. ) Central phosphate becomes permanently bound to the serine
  3. ) Blocks Ach from interacting with enzyme (AchE)
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16
Q

Once AchE is irreversibly blocked: (4)

A
  1. ) Ach levels remain high in synapse
  2. ) Continued stimulation of muscle contraction
  3. ) Respiratory muscles won’t relax
  4. ) Death by asphyxiation or blocking normal neural function
17
Q

Irreversible inhibition special category =

A

Suicide inhibitors

18
Q 
(Irreversible Inhibitors)
(Suicide inhibitors)
1.) Involves...
2.) Upon interaction with the enzyme...
3.) It then forms...
4.) Thus, the enzyme...
A
  1. ) …a compound which resembles the normal substrate for an enzyme (substrate analogue)
  2. ) …it is modified by the enzyme to produce a reactive group
  3. ) …a covalent bond and a stable inhibitor-enzyme complex, thus permanently inactivates the enzyme.
  4. ) …essentially synthesizes it’s own inhibitor
19
Q

(Irreversible Inhibitors)
(Suicide inhibitors) 2 examples:
& what they inhibit

A
  1. ) Penicillin: inhibits DD-transpeptidase

2. ) AZT: inhibits HIV-1 reverse transcriptase

20
Q

(Irreversible Inhibitors)
(Suicide inhibitors)
Penicillin: Mechanism of Action (5)

**Ultimately…

A
  1. ) The bacterial cell wall (peptidoglycan) consists of cross-linked strands of NAG and NAM (sugars). The cross-links occur at peptide chains (bound to the sugars)
  2. ) The PBP (Penicillin Binding Protein, DD-transpeptidase) is the enzyme responsible for forming the cross-links.
  3. ) The PBP dissociates from the wall once the cross-link has been formed.
  4. ) Penicillin enters the active site of the PBP.
  5. ) The beta-lactam ring (represented here as the top of the “P” representing penicillin) is altered by the PBP and penicillin covalently binds to the PBP, permanently blocking its active site.

**Ultimately, the bacterial cell wall is weakened. The bacteria dies due to osmotic pressures that cause cytolysis.

21
Q

(Reversible Inhibitors)
are not…
following inhibition…

A

…permanent

…if the inhibitor is removed, then activity will return to normal

22
Q

(Reversible Inhibitors)

4 main types:

A
  1. ) Competitive
  2. ) Uncompetitive
  3. ) Mixed
  4. ) Noncompetitive
23
Q 
(Reversible Inhibitors)
(Competitive Inhibition)
Usually involve...
Inhibitor has...
Inhibitor binds at...
A

…a substrate analogue
…a similar structure to the substrate
…active site (substrate binding pocket)

24
Q

(Reversible Inhibitors)
Competitive Inhibitors and substrate both…
Binding of a competitive inhibitor prevents…

A

…bind to the active site of the enzyme

…substrate binding, thereby inhibiting enzyme activity

25
Q

(Reversible Inhibitors)
Competitive inhibitors may or may not…
If it reacts…

A

…react as a substrate

…it will do so very slowly

26
Q

(Reversible Inhibitors)

Scientists can use competitive inhibitors to…

A

…gain info about the active site through comparison of structures

27
Q 
(Reversible Inhibitors)
Competitive Inhibition Effects:
1.) The amount of enzyme...
2.) However, the inhibitor and substrate...
3.) Thus...
A
  1. ) …that is inhibited by the inhibitor will depend on the concentration of the inhibitor (more inhibitor → more inhibition)
  2. ) …compete for the same active site
  3. ) …adding more substrate will reverse the level of inhibition
28
Q 
(Reversible Inhibitors)
(Competitive Inhibition)
- Inhibitor (I) and Substrate (S) compete for...
- Increasing [I] → ...
- Which...
A

… Enzyme (E)
… → Increases [EI]
…reduces [E] available for substrate binding

29
Q

Competitive Inhibition Effects on Kinetics: & Why?

  1. ) Vmax:
  2. ) Km:
  3. ) Slope:
A
  1. ) Vmax was unchanged (Y-intercept unchanged)
    - If you add more substrate, eventually it out competes the inhibitor for the enzyme (at very high substrate concentrations the enzyme can still achieve true Vmax)
  2. ) Km increases with increasing [I]
    - In the presence of the inhibitor, the E needs a higher [S] to reach ½ Vmax
  3. ) Slope increases
    - ratio of Km/Vmax is increased, due to increased Km
30
Q 
(Reversible Inhibitors)
(Uncompetitive Inhibition)
- Inhibitor binds to...
- Inhibitor does not need...
- Distorts...
- Increasing [S]...
- How often in nature?
A
  • …enzyme-substrate complex, not to free enzyme
  • …to resemble substrate
  • …active site; prevents reaction from occurring
  • …does not change binding of inhibitor to ES (uncompetitive)
    • Vmax is affected by [I]
  • Rare in nature
31
Q

Uncompetitive Inhibition Effects on Kinetics: & Why?

  1. ) Vmax:
  2. ) Km:
  3. ) Slope:
A
  1. ) Vmax is decreased
    - Inhibitor slows the rate at which the ES can form product
  2. ) Km is decreased
    - (think of Km as a dissociation constant for E & S)
    - Because the inhibitor only binds the ES complex (effectively decreasing the [ES]), the equilibrium actually shifts to replace the lowered ES complex, thereby increasing the apparent affinity of S for E (lower Km).
  3. ) Slope is unchanged
    - Both Vmax and Km are decreased in proportion
    - Km/Vmax is unchanged
32
Q

(Reversible Inhibitors)
(Mixed Inhibition)
- Inhibitor binds to…
- Which results in…

A
  • …either E or ES (not at active site)

- a combination of effects

33
Q

Mixed Inhibition Effects on Kinetics: & Why?

  1. ) Vmax:
  2. ) Km:
  3. ) Slope:
A
  1. ) Vmax is lowered (y-int increases)
    - Decreases the rate of product formation
  2. ) Km is raised (x-int increases)
    - Need a higher [S] to reach ½ Vmax
  3. ) Slope is increased
    - Ratio of Km/Vmax is changed
34
Q 
(Reversible Inhibitors)
(Noncompetitive Inhibition)
- Essentially a...
- Inhibitor binds...
- Effectively...
- Also, ...
- ...which will...
A
  • …special case of mixed inhibition
  • …the “free” enzyme away from the active site
  • …removes enzyme from functional pool
  • …“ties up” some of the enzyme
  • …prevent some proportion of the E to participate in the reaction
35
Q

Noncompetitive Inhibition Effects on Kinetics: & Why?

  1. ) Vmax:
  2. ) Km:
A
  1. ) Vmax is decreased
    - Less functional enzyme in presence of inhibitor, therefore the maximum rate of product formation is reduced
  2. ) Km is unchanged
    - Because the inhibitor essentially reduces the amount of functional E to a lower Vmax, the [S] at ½ of the ‘new’ Vmax (i.e., the ‘new’ Km) is unchanged
36
Q

Draw all 4 reversible inhibitor kinetics graphs!!

  • competitive
  • uncompetitive
  • mixed
  • noncompetitive
A

do it plzzzzzzz don’t you want an A????

37
Q

Summary of Reversible Inhibition: (Vmax & Km)

  1. ) competitive
  2. ) uncompetitive
  3. ) mixed
  4. ) noncompetitive
A 
1.) Competitive
   VMax: same
   Km: Increased
2.) Uncompetitive
   VMax: decreased
   Km: decreased
3.) Mixed
   VMax: decreased
   Km: Increased
4.) Noncompetitive
   VMax: decreased
   Km: same
38
Q

M-M kinetics are based on…

A

a simple reaction scheme:

E + S ↔ ES ↔ E + P

39
Q

Other reaction mechanisms exist (2 substrates): (3)

& draw them!!!

A
  1. ) Random
  2. ) Ordered
  3. ) Ping-pong

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