6. Enzymes & Drugs Flashcards
What are inhibitors?
Inhibitors are small molecules that interfere with catalysis, slowing, or halting enzymatic reactions.
What are the three types of inhibition?
- Reversible, competitive
- Reversible, uncompetitive
- Irreversible
competitive inhibition
- 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.
What are transition state analogs?
Transition state analogs are particularly effective competitive inhibitors because they bind stronger to the enzyme rather than the substrate.
How can enzymatic products control enzymatic activity?
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.
What is the relationship of competitive inhibition and the concentration of free enzyme?
A competitive inhibition reduces the concentration of free enzyme available for substrate binding.
Draw a diagram of normal enzyme-substrate binding and reversible, uncompetitive inhibition.
Write the enzymatic equation for reversible, uncompetitive inhibition.
What changes must occur to the Michaelis-Menten equation to account for competitive inhibition?
T/F: The degree of competitive inhibition varies with the fraction of enzyme that has bound inhibitor.
True.
T/F: The presence of competitive [I] makes [S] appear to be greater than what it really is.
False. The presence of [I] makes [S] appear to be less than what it really is.
In regards to competitive inhibition, what does α indicate?
α is a factor by which [S] must be increased to overcome a competitive inhibitor.
T/F: A competitve inhibitor affects the enzyme’s turnover number.
False. The inhibitor does not affect the enzyme’s turnover number.
As [S] reaches infinity, V0 approaches Vmax for any concentration of inhibitor.
How does reversible, competitive inhibition affect Vmax and Km?
Show what this would look like on a Lineweaver-Burk function.
- 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.
How does reversible, competitive inhibition affect Vmax and Km?
Show what this would look like on a Michaelis-Menten function.
- 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.
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.
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.
Describe the mechanism of action for methotrexate.
Methotrexate is a competitive inhibitor of DHFR, which prevents the conversion of DHF to THF.
Which enzymes are inhibited in HIV drug regiments?
Explain.
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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.
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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.
Describe the mechanism of action of reverse transcriptase inhibition.
- 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!
Describe the mechanism of action of HIV protease.
- 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.
What is the mechanism of action of peptidomimetic drugs?
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.
What is a proteasome?
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.
What is Bortezomib and MG132?
How does it generally work to inhibit proteasomes?
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.
What occurs at the β subunit of proteasome inhibitors? That is, what amino acid is involved?
The active sites use the hydroxyl group of the N-terminal threonine residues to attack peptide bonds.
Describe how Bortezomic specifically affects the active site of proteasomes.
Bortezomib form an adducts with the threonine that mimic the transition state intermediate during peptide cleavage.
Describe the mechanism of action of allopurinol to treat gout?
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.
Draw a diagram of normal enzyme-substrate binding and reversible, competitive inhibition.
Write the enzymatic equation for reversible, competitive inhibition.
How does reversible, uncompetitive inhibition affect Vmax and Km?
Show what this would look like on a Lineweaver-Burk function.
- Apparent Vmax = Vmax/α’
- Apparent Km = Km/α’
- Apparent Vmax lower, apparent Km decreases
Increasing [S] will not be able to overcome the inhibitor. However, enzyme will still be effective at low [S].
How does reversible, uncompetitive inhibition affect Vmax and Km?
Show what this would look like on a Michaelis-Menten function.
- 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.
Describe the mechanism of action of non-nucleoside reverse transcriptase inhibitors (NNRTIs)?
NNRTI locks the reverse transcriptase in one conformation.
Draw a diagram of normal enzyme-substrate binding and mixed inhibition.
Write the enzymatic equation for mixed inhibition.
Mixed inhibition involves inhibitor binding to both the free enzyme and the enzyme-substrate complex.
How does mixed affect Vmax and Km?
Show what this would look like on a Lineweaver-Burk function.
- Apparent Vmax = Vmax/α’
- Apparent Km = αKm/α’
Increasing [S] will not be able to overcome the inhibitor. However, enzyme will still be effective at low [S].
Describe mixed noncompetitive inhibition.
How does this affect Vmax and Km.
Noncompetitive inhibition: When the inhibitor binds to E and ES with the same binding affinity (KI = KI’ or α = α’)
- Apparent Vmax = Vmax/α
- Apparent Km = Km
Describe irreversible inhibition.
- 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.
What is the mechanism of action of penicillin?
- 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.
Describe the action of glycopeptide transpeptidase.
- 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.
Describe the mechanism of fluorouracil as an anticancer drug.
- 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.
What is the mechanism of carfilzomib?
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).
What is the mechanism of action of NSAIDs?
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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.
Describe the mechanism of action of aspirin at the active site of PGH2 synthase-1.
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.
What two groups are drug-metabolizing enzymes categorized within?
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.
Describe the drug-drug interaction between midazolam and clarithromycin.
What type of inhibition is taking place?
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.
Describe the drug-drug interaction between theophylline and enoxacin.
What type of inhibition is taking place?
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
