6. Enzymes & Drugs

Question 1

What are inhibitors?

Answer 1

Inhibitors are small molecules that interfere with catalysis, slowing, or halting enzymatic reactions.

Question 2

What are the three types of inhibition?

Answer 2

1. Reversible, competitive
2. Reversible, uncompetitive
3. Irreversible

Question 3

competitive inhibition

Answer 3

• Competitive inhibition involves inhibitor binding at an enzyme’s substrate binding site.
• A substance that directly competes with a normal substrate for the enzyme’s binding site is known as a competitive inhibitor.
• Competitive inhibitor resembles normal substrate.

Ex. Best examples are transition state analogs.

Question 4

What are transition state analogs?

Answer 4

Transition state analogs are particularly effective competitive inhibitors because they bind stronger to the enzyme rather than the substrate.

Question 5

How can enzymatic products control enzymatic activity?

Answer 5

Products of enzymatic reactions, which are necessarily able to bind to the enzyme’s active site, may accumulate and compete with substrates for binding to the enzyme.

This is one mechanism through which the cell controls activity of its enzymes.

Question 6

What is the relationship of competitive inhibition and the concentration of free enzyme?

Answer 6

A competitive inhibition reduces the concentration of free enzyme available for substrate binding.

Question 7

Draw a diagram of normal enzyme-substrate binding and reversible, uncompetitive inhibition.

Write the enzymatic equation for reversible, uncompetitive inhibition.

Answer 7

Question 8

What changes must occur to the Michaelis-Menten equation to account for competitive inhibition?

Answer 8

Question 9

T/F: The degree of competitive inhibition varies with the fraction of enzyme that has bound inhibitor.

Answer 9

True.

Question 10

T/F: The presence of competitive [I] makes [S] appear to be greater than what it really is.

Answer 10

False. The presence of [I] makes [S] appear to be less than what it really is.

Question 11

In regards to competitive inhibition, what does α indicate?

Answer 11

α is a factor by which [S] must be increased to overcome a competitive inhibitor.

Question 12

T/F: A competitve inhibitor affects the enzyme’s turnover number.

Answer 12

False. The inhibitor does not affect the enzyme’s turnover number.

As [S] reaches infinity, V₀ approaches V_max for any concentration of inhibitor.

Question 13

How does reversible, competitive inhibition affect V_max and K_m?

Show what this would look like on a Lineweaver-Burk function.

Answer 13

• Apparent V_max same
• Apparent K_m increased

This indicates that increasing [S] can overcome the competitive inhibitor as V_max is approached.

However, the enzyme may not be as effective at low [S] when exposed to competitive inhibitor.

Question 14

How does reversible, competitive inhibition affect V_max and K_m?

Show what this would look like on a Michaelis-Menten function.

Answer 14

• Apparent V_max same
• Apparent K_m increased

This indicates that increasing [S] can overcome the competitive inhibitor as Vmax is approached.

However, the enzyme may not be as effective at low [S] when exposed to competitive inhibitor.

Question 15

Cancer cells have greater requirements for nucleotides as precursors of DNA and RNA, and are consequently more sensitive than normal cells to inhibitors of nucleotide biosynthesis.

Therefore, what are two enzymes that can be inhibited to provide an effective therapy for cancer? Name their substrates.

Answer 15

Thymidylate synthase and dihydrofolate reductase (DHFR) are the two enzymes that provide the only cellular pathway for thymidine synthesis.

• Thymidylate synthase binds to THF
• DHFR binds to dihydrofolate DHF.

DHFR converts DHF to THF.

Question 16

Describe the mechanism of action for methotrexate.

Answer 16

Methotrexate is a competitive inhibitor of DHFR, which prevents the conversion of DHF to THF.

Question 17

Which enzymes are inhibited in HIV drug regiments?

Explain.

Answer 17

1. Reverse transcriptase
   
   • HIV is a retrovirus. They possess an RNA genome and an enzyme, reverse transcriptase, capable of using RNA to direct the synthesis of a complimentary DNA. The duplex DNA is then inserted into a chromosome in the nucleus of the host cell.
2. HIV protease
   
   • Viral genome can be transcribed back into RNA, which can then be translated into proteins to construct new virus particles. Most of the viral genes are translated into large polyproteins, which are cut by the HIV protease into the individual proteins needed to make the virus.

Question 18

Describe the mechanism of action of reverse transcriptase inhibition.

Answer 18

• Archetype is AZT (3’-azido-3’-deoxythymidine; Zidovudine): taken up by cells, phosphorylated and incorporated into DNA but does not support chain elongation because it lacks 3’ OH.
• Cellular polymerases have low affinity for this drug but reverse transcriptase has high affinity!

Question 19

Describe the mechanism of action of HIV protease.

Answer 19

• HIV protease is a homodimer of 99-residue subunits.
• It is an aspartyl protease, because aspartic acids act as catalysts at its active site.
• Cleaves Phe-Pro or Tyr-Pro peptide bond, through a tetrahedral transition state (highlighted in pink).
• General acid (proton donor) - base (proton acceptor) catalysis by two active-site Asp residues (from different subunits), facilitating the attack of water on the peptide bond.

Question 20

What is the mechanism of action of peptidomimetic drugs?

Answer 20

Peptidomimetic drugs (e.g., ritonavir and saquinavir) contain bulky groups that bind to the active site of HIV protease, mimicking the tetrahedral transition state of viral polyproteins.

Question 21

What is a proteasome?

Answer 21

The proteasome is a large protein complex responsible for degradation of intracellular proteins. Polymerization of ubiquitin, a key molecule known to work in concert with the proteasome, serves as a degradation signal for numerous target proteins.

Question 22

What is Bortezomib and MG132?

How does it generally work to inhibit proteasomes?

Answer 22

Bortezomib and MG132 are competitive, reversible proteasome inhibitors used as an anticancer drug.

There are three types of proteolytic sites in the 20S proteasome’s central chamber, and each β ring contains three active sites. Bortezomib and MG132 act primarily on the chymotrypsin-like site in the β subunit but also inhibit the caspase-like site at high concentrations.

Question 23

What occurs at the β subunit of proteasome inhibitors? That is, what amino acid is involved?

Answer 23

The active sites use the hydroxyl group of the N-terminal threonine residues to attack peptide bonds.

Question 24

Describe how Bortezomic specifically affects the active site of proteasomes.

Answer 24

Bortezomib form an adducts with the threonine that mimic the transition state intermediate during peptide cleavage.

Question 25

Describe the pathophysiology of gout. What enzyme causes gout?

Answer 25

Gout is a disease of the joints caused by an **elevated concentrations of uric acid in the blood and tissues.** Gout often involves underexcretion of urine by kidneys. The joints become inflamed, painful, and arthritic, owing to abnormal deposition of sodium urate crystals. The kidneys are also affected, as excess uric acid is deposited in the kidney tubules. The enzyme **xanthine oxidase catalyzes the conversion of purines to uric acid.** Hencer, it is a target for treating gout.

Question 26

Describe the mechanism of action of allopurinol to treat gout?

Answer 26

**When xanthine oxidase is inhibited, the excreted products of purine metabolism are xanthine and hypoxanthine, which are more water soluble** than uric acid and less likely to form crystalline deposits. Hypoxanthine is the normal substrate of xanthine oxidase. Only a slight alteration in the structure yields allopurinol. At the active site, allopurinol is converted to oxypurinol, a strong competitive inhibitor that remains tightly bound to the enzyme.

Question 27

Where does an uncompetitive inhibitor bind?

Answer 27

* **Uncompetitive inhibitor binds to the enzyme-substrate complex, but not at the substrate binding site as with competitive inhibitor.** * **Upon binding, uncompetitive inhibitors induce a conformational change which makes the enzyme inactive.** * Uncompetitive inhibitors need not to resemble the substrate but distort the active site, thereby rendering the enzyme catalytically inactive.

Question 28

What occurs to an enzyme exposed to uncompetitive inhibitors exposed to low and high [S]?

Answer 28

Increasing [S] does not allow the enzyme to overcome the inhibitor. In fact, it does not reverse the effect of the uncompetitive inhibitor. However, the enzyme will still be effective at low [S] even when exposed to competitive inhibitors.

Question 29

Draw a diagram of normal enzyme-substrate binding and reversible, competitive inhibition. Write the enzymatic equation for reversible, competitive inhibition.

Answer 29

Question 30

How does reversible, uncompetitive inhibition affect V_max and K_m? Show what this would look like on a Lineweaver-Burk function.

Answer 30

* Apparent V_max = V_max/α' * Apparent K_m = K_m/α' * Apparent V_max lower, apparent K_m decreases Increasing [S] will not be able to overcome the inhibitor. However, enzyme will still be effective at low [S].

Question 31

How does reversible, uncompetitive inhibition affect V_max and K_m? Show what this would look like on a Michaelis-Menten function.

Answer 31

* Apparent Vmax same * Apparent Km increased This indicates that increasing [S] can overcome the competitive inhibitor as Vmax is approached. However, the enzyme may not be as effective at low [S] when exposed to competitive inhibitor.

Question 32

Describe the mechanism of action of non-nucleoside reverse transcriptase inhibitors (NNRTIs)?

Answer 32

NNRTI locks the reverse transcriptase in one conformation.

Question 33

Draw a diagram of normal enzyme-substrate binding and mixed inhibition. Write the enzymatic equation for mixed inhibition.

Answer 33

Mixed inhibition involves inhibitor binding to both the free enzyme and the enzyme-substrate complex.

Question 34

How does mixed affect V_max and K_m? Show what this would look like on a Lineweaver-Burk function.

Answer 34

* Apparent V_max = V_max/α' * Apparent K_m = αK_m/α' Increasing [S] will not be able to overcome the inhibitor. However, enzyme will still be effective at low [S].

Question 35

Describe mixed noncompetitive inhibition. How does this affect V_max and K_m.

Answer 35

Noncompetitive inhibition: When the inhibitor binds to E and ES with the same binding affinity (K_I = K_I’ or α = α’) * **Apparent V_max = V_max/α** * **Apparent K_m = K_m**

Question 36

Describe irreversible inhibition.

Answer 36

* **Irreversible inhibitors bind covalently to the enzyme (most common), or destroy a functional group that is required for the enzyme’s activity, or form a highly stable non-covalent association.** * The kinetics of an inactivator or irreversible inhibitor resembles that of a mixed inhibitor because the inactivator reduces the concentration of functional enzyme at all substrate concentrations.

Question 37

What is the mechanism of action of penicillin?

Answer 37

* Penicillin interferes with the synthesis of peptidoglycan, the major component of the rigid cell wall that protects bacteria from osmotic lysis. * Peptidoglycan formation is catalyzed by the enzyme glycopeptide transpeptidase. * Therefore, penicillin mimics the the normal substrate that activates glycopeptide transpeptidase cataylsis.

Question 38

Describe the action of glycopeptide transpeptidase.

Answer 38

* Bacterial cell walls contain **D-amino acids**, which **form cross-links** by a mechanism different from that used to synthesize normal proteins. * The transpeptidase normally forms an acyl intermediate with the penultimate D-alanine residue of the **D-Ala-D-Ala** peptide. * This covalent acyl-enzyme intermediate then reacts with the amino group of the terminal glycine in another peptide to form the cross-link.

Question 39

Describe the mechanism of fluorouracil as an anticancer drug.

Answer 39

* Thymidylate synthase converts deoxy uracil monophosphate (dUMP) to deoxy thymidine monophosphate (dTMP). In RNA, uracil is the pyrimidine nucleobase, whereas in DNA, its derivative thymine is the nucleobase. * Fluorouracil converts to FdUMP in the cell, which acts as an irreversible inhibitor of thymidylate synthase. * It forms a dead-end covalent complex of fluorouracil with thymidylate synthase.

Question 40

What is the mechanism of carfilzomib?

Answer 40

Carfilzomib is an anti-cancer drug that is a irreversible proteasome inhibitor. It is an analog of epoxomicin. Epoxomicin inhibits the proteasome by forming a morpholine adduct with a threonine residue at the N-terminus of b5 subunits (atoms from epoxomicin in blue; atoms from the proteasome in red).

Question 41

What is the mechanism of action of NSAIDs?

Answer 41

* **Inhibit the cyclooxygenase (COX) activity of the enzyme prostaglandin H₂ synthase-1.** * Prostaglandin H₂ (PGH2) promotes inflammation. * The enzyme catalyzes the conversion of arachidonic acid into PGH2 in two steps: (1) a cyclooxygenase reaction, and (2) a peroxidase reaction.

Question 42

Describe the mechanism of action of aspirin at the active site of PGH2 synthase-1.

Answer 42

NSAIDS block the hydrophobic channel and prevent PGH2 synthesis by inhibiting the COX activity of the enzyme. Aspirin is an irreversible inhibitor of PGH2 synthase-1. It covalently modifies (acetylates) specific Ser-OH group in the channel through which substrate (arachidonic acid) must pass to reach the active site.

Question 43

Describe the mechanism of action of ibuprofen, acetaminophen, and naproxen at the active site of PGH2 synthase.

Answer 43

* Reversible, competitive inhibitors of COX activity of PGH2 synthase * Reversibly bind in channel through which substrate must access enzyme active site, so these drugs act as competitive inhibitors by preventing substrate binding, even though they don’t bind in the active site.

Question 44

What two groups are drug-metabolizing enzymes categorized within?

Answer 44

Drug metabolizing enzymes have been grouped into those that carry out: * **Phase 1 reactions**, which include oxidation, reduction, or hydrolytic reactions * **Pphase 2 reactions**, in which enzymes catalyze the conjugation of the substrate (the phase 1 product) with a second molecule.

Question 45

What can occur when a drug-metabolizing enzyme is inhibited? What are the two kinds of inhibition that can take place in this drug-drug interaction?

Answer 45

Inhibition of a drug-metabolizing enzyme by one drug leads to increased levels of drugs metabolized by the same enzyme. This can often leads to decreased levels of drug required, or drug toxicity. * Competitive inhibition * Uncompetitive/noncompetitive inhibition

Question 46

Describe the drug-drug interaction between midazolam and clarithromycin. What type of inhibition is taking place?

Answer 46

Non-competitive inhibition * Clarithromycin binds to cytochrome P450 at a site other than the active site. * Binding of clarithromycin inactivates the enzyme. * Time it takes to wear off is guided by the half-life of the inhibitor and the time it takes to synthesize new enzyme.

Question 47

Describe the drug-drug interaction between theophylline and enoxacin. What type of inhibition is taking place?

Answer 47

Competitive inhibition * Both are metabolized by cytochrome P450 and bind to the same active site. * Theophylline plasma concentration increases with the administration of enoxacin. * Theophylline plasma concentration reverses back to its normal levels after the enoxacin stopped. Time it takes to wear off is guided by the half-life of the inhibitor. * Increased enoxacin levels lead to increased plasma theophylline concentration