Lec 4: Enzymes (Pt. 2)

Question 1

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

Answer 1

…the Km and Vmax when plotted as a hyperbolic function

…the Km and Vmax with the Lineweaver-Burke Plot

Question 2

Enzyme Inhibitors =

Answer 2

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

Question 3

Why have enzyme inhibitors? (3)

Answer 3

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

Question 4

Many ________ ______ can act as…

Answer 4

chemical agents

…inhibitors (even ATP, which inhibits PFK)

Question 5

Each enzyme has

Answer 5

specific inhibitors (specificity)

Question 6

(Alcohols) partake in…

and can…

Answer 6

…hydrogen bonding

…dehydrate tissue

Question 7

Reducing agents can directly…

therefore…

Answer 7

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

Question 8

Inhibitors: 2 Main Categories

Answer 8

1. ) Irreversible Inhibitors

2. ) Reversible Inhibitors

Question 9

(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, …

Answer 9

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.

Question 10

Irreversible Inhibitors: (3)

& examples:

Answer 10

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

Question 11

(Irreversible Inhibitors)
Organophosphates inhibit…
by…

Answer 11

…the active site of acetylcholinesterase

…reacting with the hydroxyl group of serine permanently modifying it

Question 12

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

Answer 12

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

Question 13

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

Answer 13

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

Question 14

Acetylcholinesterase (AchE) is…

and prevents…

Answer 14

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

Question 15

(DFP inhibition of Acetylcholinesterase) Steps: (3)

Answer 15

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

Question 16

Once AchE is irreversibly blocked: (4)

Answer 16

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

Question 17

Irreversible inhibition special category =

Answer 17

Suicide inhibitors

Question 18

(Irreversible Inhibitors)
(Suicide inhibitors)
1.) Involves...
2.) Upon interaction with the enzyme...
3.) It then forms...
4.) Thus, the enzyme...

Answer 18

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

Question 19

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

Answer 19

1. ) Penicillin: inhibits DD-transpeptidase

2. ) AZT: inhibits HIV-1 reverse transcriptase

Question 20

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

**Ultimately…

Answer 20

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.

Question 21

(Reversible Inhibitors)
are not…
following inhibition…

Answer 21

…permanent

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

Question 22

(Reversible Inhibitors)

4 main types:

Answer 22

1. ) Competitive
2. ) Uncompetitive
3. ) Mixed
4. ) Noncompetitive

Question 23

(Reversible Inhibitors)
(Competitive Inhibition)
Usually involve...
Inhibitor has...
Inhibitor binds at...

Answer 23

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

Question 24

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

Answer 24

…bind to the active site of the enzyme

…substrate binding, thereby inhibiting enzyme activity

Question 25

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

Answer 25

...react as a substrate | ...it will do so very slowly

Question 26

(Reversible Inhibitors) | Scientists can use competitive inhibitors to...

Answer 26

...gain info about the active site through comparison of structures

Question 27

``` (Reversible Inhibitors) Competitive Inhibition Effects: 1.) The amount of enzyme... 2.) However, the inhibitor and substrate... 3.) Thus... ```

Answer 27

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

Question 28

``` (Reversible Inhibitors) (Competitive Inhibition) - Inhibitor (I) and Substrate (S) compete for... - Increasing [I] → ... - Which... ```

Answer 28

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

Question 29

Competitive Inhibition Effects on Kinetics: & Why? 1. ) Vmax: 2. ) Km: 3. ) Slope:

Answer 29

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

Question 30

``` (Reversible Inhibitors) (Uncompetitive Inhibition) - Inhibitor binds to... - Inhibitor does not need... - Distorts... - Increasing [S]... - How often in nature? ```

Answer 30

- ...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

Question 31

Uncompetitive Inhibition Effects on Kinetics: & Why? 1. ) Vmax: 2. ) Km: 3. ) Slope:

Answer 31

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

Question 32

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

Answer 32

- ...either E or ES (not at active site) | - a combination of effects

Question 33

Mixed Inhibition Effects on Kinetics: & Why? 1. ) Vmax: 2. ) Km: 3. ) Slope:

Answer 33

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

Question 34

``` (Reversible Inhibitors) (Noncompetitive Inhibition) - Essentially a... - Inhibitor binds... - Effectively... - Also, ... - ...which will... ```

Answer 34

- ...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

Question 35

Noncompetitive Inhibition Effects on Kinetics: & Why? 1. ) Vmax: 2. ) Km:

Answer 35

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

Question 36

Draw all 4 reversible inhibitor kinetics graphs!! - competitive - uncompetitive - mixed - noncompetitive

Answer 36

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

Question 37

Summary of Reversible Inhibition: (Vmax & Km) 1. ) competitive 2. ) uncompetitive 3. ) mixed 4. ) noncompetitive

Answer 37

``` 1.) Competitive VMax: same Km: Increased 2.) Uncompetitive VMax: decreased Km: decreased 3.) Mixed VMax: decreased Km: Increased 4.) Noncompetitive VMax: decreased Km: same ```

Question 38

M-M kinetics are based on...

Answer 38

a simple reaction scheme: | E + S ↔ ES ↔ E + P

Question 39

Other reaction mechanisms exist (2 substrates): (3) | & draw them!!!

Answer 39

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