Medicine - Enzyme Inhibitors 2 - Suicide Inactivators And Transition State Analogies

Question 1

Describe the use of 5-flourouracil as a suicide inhibitor

Answer 1

5-flourouracil is converted in the body to the fluorinated analogue of 2’-deoxyuridine monophase (FdUMP)
This suicide inactivator is used to inhibit the enzyme thymidylate synthase, which is involved in DNA biosynthesis

Question 2

Define suicide inactivator

Answer 2

Agents which are converted to irreversible inhibitors by the enzyme-catalysed reaction
Not active until it is bound to the active site (chemically)
React with target enzyme once contact is formed

Question 3

What is the mode of interactions of suicide inactivators within an enzyme?
What is the effect of increasing the substrate?

Answer 3

At the enzyme active site these inhibitors form a permanent complex with an enzyme via formation of covalent bonds with certain amino acids in the active site of an enzyme
Consequently, they cannot be displaced by the addition of excess substrate
Therefore, they are irreversible, non-competitive enzyme inhibitors

Question 4

Diagram/series of reactions for suicide inactivators

Answer 4

E+I⇌(inhibitor binds)EI(normal catalytic reaction starts at t active site⇌E…I (reactive species generated) and forms E-I (inactivated enzyme)

NB: enzyme treats the inhibitor as if it is a “normal” substrate/substance

Question 5

Mechanism-based inhibitors depend on…

Answer 5

Depend on the specific mechanism of catalysis of a particular target enzyme

Question 6

Advantages of mechanism-based enzyme inhibitors

Answer 6

• Absolute specificity: they depend upon substrate-like binding recognition and upon specific mechanism of catalysis in the active site of a particular enzyme
• High efficiency of enzyme inhibition (covalent complex)

Question 7

What is Tienilic acid?

Answer 7

Drug marked as a diuretic agent
Suicide inactivator
Withdrawn due to interaction with cytochrome P450 enzymes in the liver, where it acts as a suicide substrate

Question 8

What helps design powerful inhibitors?

Answer 8

Understanding of enzyme mechanism of reaction helps to design more powerful inhibitors

Question 9

Transition state analogues

Answer 9

It is possible to design inhibitors that bing extremely strongly to the active site.
One approach to achieve this is to design a drug that resembles the transition state of the substrate in the active site
BUT
Drug design can be based on reaction intermediates which are closer in character to transition states than substrates or products

Question 10

Define transition state

Answer 10

High energy, transient species

Cannot be isolated or synthesised

Question 11

What are transition state analogues designed to mimic?

How strongly do they bind?

Answer 11

The transition state of the enzyme-catalysed reaction

They are more likely to bind more strongly than drugs mimicking the substrate or product

Question 12

Penicillins are what type of enzyme inhibitor?

Is there an alternative theory?

Answer 12

Penicillins are irreversible suicide inhibitors of transpeptidase
Penicillin becomes covalently linked to the enzyme’s active site
Alternative theory: Penicillin mimics D-Ala-D-Ala

Question 13

How does penicillin resistance occur?

Answer 13

Via beta-lactamases
These enzymes inactivate penicillins by opening beta-lactam rings
This allows bacteria to become resistant to penicillin

Question 14

Beta-lactamase mechanism of action:

Answer 14

Mechanism of action for beta-lactamases is identical to the mechanism of inhibition of the target enzyme (i.e.transpeptidase)
But the product is removed effectively fro the lactamase active site

Question 15

How can you overcome penicillin resistance?

Answer 15

Use clavulanic acid

Question 16

What is Clavulanic acid?

Answer 16

-weak, unimportant antibacterial activity
BUT
-Powerful irreversible suicide inhibitor of beta-lactamase (enzyme that causes bacterial resistance to penicillins)
-used as a guard drug to ampicillin

Question 17

Where does clavulanic acid come from

Answer 17

Isolated from

Streptomyces clavoligenus

Question 18

Augmentin=

Answer 18

Ampicillin+clavulanic acid

Allows less ampicillin per dose and increased activity spectrum

Question 19

Timentin=

Answer 19

Ticarcillin + clavulanic acid

Question 20

Why should a transition state be bound more strongly to an enzyme that a substrate or product?

Answer 20

It is ONLY HYPOTHESIS:

• it is proposed that the binding interactions between an enzyme and a substate are optimal during the transition state of the enzyme-catalysed reaction
• this is a reasonable proposition since the speed and effectiveness of a catalysed reaction is crucially dependent on how much the catalyst stabilises the transition state

Question 21

How alter the transition state to make the reaction occur more easily and make the enzyme more effective?

Answer 21

The more stable the transition state, the easier the reaaction will occur and the more effective the enzyme will be as a catalyst
Therefore
It is more important that enzymes form their strongest interactions with ‘guest molecules’ at the transition state of the reaction, rather than the substrate or the product

Question 22

Strong interactions with substrate or product would be…

Answer 22

Detrimental because it would result in slow “turnover” with substrate and product spending too much time in the active site

Question 23

Name an enzyme of a transition state inhibitor and describe how it works

Answer 23

Renin inhibitors (Antihypertensives)

Renin is a protease enzymes which is responsible for hydrolysing a specific peptide bond in the protein angiotensinogen
This converts angiotensinogen into angiotensin I, which then (via an ACE enzyme) is converted into angiotensin II

Angiotensin II is a vasoconstrictor and constricts blood vessels, thus raises blood pressure. Renin blocks synthesis of of angiotensin I and thus, angiotensin II

Question 24

Describe how renin works

Answer 24

Renin contains two aspartyl residues and a bridging water in the active site
The first stage involves formation of a tetrahedral intermediate via processing through a high energy transition state
The two aspartyl resides are involved in the enzyme-catalysed reaction and the tetrahedral intermediate is involved.

Question 25

What is Aliskiren? What does it do?

Answer 25

Aliskiren is a renin inhibitor

• it contains a hydroxyethylene transition state mimic
• it therefore mimic the tetrahedral geometry of the usual reaction intermediate when renin catalyses angiotensinogen
• it mimics one of the hydroxyl groups (binding group)
• it is stable, no leaving group present

Question 26

Tetrahedral geometry of transition states is in what enzymes?

Answer 26

Statins
Renin
ACE inhibitors
Protease inhibitors

Question 27

Is transition-state inhibition usually reversible or irreversible?

Answer 27

-Reversible inhibition, unless very tight binding, as no covalent bond formed

Question 28

Criteria for transition-state inhibition?

Answer 28

• Reversible (unless v.tight binding)
• Close structural resemblance to postulated transition state
• Tighter binding to the enzyme than substrate binding
• often seen as competitive inhibition as active site is targeted and increase in substrate concentration may displace the inhibitor. This may mean that the transition state analogue forces the enzyme to adopt a new conformation
• sometimes inhibition is slow (minutes to hours) as conformation is crucial

Question 29

Are transition state inhibitors competitive or non-competitive

Answer 29

Often seen as competitive as active site is targeted
Increase in substrate concentration may displace the inhibitor
This may mean that the transition state analogue forces the enzyme to adopt a new conformation