2-8 Enzyme Mechanism and Therapeutic Inhibition of Enzymes

Question 1

What are the 6 classes of enzymes?

Answer 1

1. Oxidoreductases catalyze e^- transfer
2. Transferases catalyze transfer of a functional group
3. Hydrolases catalyze cleavage of bonds by water
4. Lyases catalyze addition of groups across a double bond or formation of a double bond
5. Isomerases catalyze isomerization of molecules
6. Ligases catalyze formation of bonds between molecules

Question 2

What are the two general strategies enzymes use to lower the transition state energy?

Answer 2

1. Direct stabilization of the transition state: enzymes bind preferentially to the transition state (enhanced “fit”) and utilize proximity and orientation effects
2. Chemical assistance at the active site: acid-base catalysis, (transient) covalent catalysis, metal ion catalysis, electrostatic (charge distribution) catalysis

Question 3

What is enzymatic suicide inhibition?

Answer 3

Suicide inhibitors bind to the active site because of resemblance to substrate. Initial steps of enzyme activity then convert them to a form capable of irreversible inhibition.

ex) nerve gases, organic fluorophosphates that irreversibly inhibit acetylcholinesterase

Question 4

How can knowledge of enzyme mechanisms be harnessed in drug design?

Answer 4

The best inhibitors often mimic the transition state conformation (enzymes tend to show preferential binding of the transition state), so the overall strategy is as follows:

• Model active site and active site conformation at transition state
• Use computer modeling to find molecules predicted to fit enzyme conformation
• Synthesize these molecules and a large number of derivatives with slight structural differences
• Test designed inhibitors for inhibition of: purified enzyme, enzyme in cells, function of target enzyme in animal models, and function of target enzyme in humans

Question 5

What are the major enzymes in the life cycle of HIV-1, and why are they significant?

Answer 5

1. Reverse transcriptase makes the first DNA strand using the single stranded RNA genome as a template, so that HIV-1 can express its viral proteins
2. Integrase catalyzes the integration of the double stranded DNA generated from the viral RNA into the host chromosomal DNA
3. HIV-1 protease cleaves the polyprotein that is the translation product of the integrated viral DNA to release individual viral proteins essential for maturation and infectivity of the virus

Inhibitors of all three of these enzymes are being used as drugs in treatment of HIV-1 infection.

Question 6

What are the characteristics of HIV-1 protease?

Answer 6

• Function: processing of viral polyprotein. Inhibition of HIV-1 protease results in the formation of immature virions that are incompetent for further infection
• Structure: aspartyl protease; homodimer, with each subunit contributing 1 Asp to the active site
• Mechanism: like other aspartyl proteases (single, tetrahedryl transition state)
• Specificity: DOES NOT have absolute substrate specificity; must cleave at several different sequences in polyprotein; large, hydrophobic, active site crevice; formation of multiple hydrophobic contacts helps dictate substrate specificity

Question 7

What are some problems with the clinical use of HIV-1 protease inhibitors?

Answer 7

• Can’t inhibit other aspartyl proteases in body
• Predominant use of substrate-based design → all inhibitors bind at the enzyme’s active site and share structural similarity
• Resistance (high error rate by reverse transcriptase + large # viral particles → viral proteins that no longer bind the inhibitor tightly)
• Pharmacokinetics (accessing reservoirs of virus; hydrophobic active site but hydrophilic delivery)
• Cost and availability
• Side effects/long-term toxicity
• Patient compliance
• When to initiate treatment

Question 8

What is the recommended drug therapy to manage AIDS?

Answer 8

• To combat resistance problems, combined drug therapy: usu. one or more reverse transcriptase inhibitors + protease inhibitor + (sometimes) another drug targeting some point in the viral life-cycle
• HAART (Highly Active Antiretroviral Therapy) or ART (Antiretroviral Therapy)
• Protease inhibitors are designed to bind at the active site of HIV protease