Lecture 3: Regulation and Inhibition of Enzymes Flashcards
___ bind substrate(s), increase rate of reaction, and are NOT consumed during reaction
Enzymes
____: Special pocket formed by protein folding that contains amino acids that participate in substrate binding and catalysis, which chemically facilitates a reaction
Active Site
_____forms upon binding of substrate to the enzyme and leads to conformation change
Enzyme-substrate (ES) complex
The ___ changes shape slightly as substrate binds
Enzyme
Kcat (_____) = molecules substrate converted to
product(s) per molecule enzyme per second
Turnover number
True or False: Enzymes have
catalytic efficiency and specificity
True
(enzymes usually interact with one or very few substrates and catalyze only
one type of reaction = specificity)
_____:non-protein molecules that enhance
enzyme activity
Co-factor/Co-enzyme
Zinc or iron is an example of a ____while small organic molecule derived from vitamins (NAD+) are examples of ____
cofactor; coenzyme
___: an active enzyme associated with its non-protein
component
____: inactive enzyme without its non-protein component
Holoenzyme
Apoenzyme
Enzymes provide a more energetically favorable path from reactants to
products by lowering the ______ barrier
activation energy
True or False; Each reaction has an activation energy barrier separating the substrates (reactants) and the products
True
____: energy difference
between reactants and transition state
Free energy of activation
_____: high-energy intermediate formed
prior to product formation
Transition state (T*)
Enzymes ↓ free energy of activation required to reach ___
Transition state (T*)
True or False: Free energy of overall reaction (ΔG) is unchanged when enzyme acts on a reaction
True
True or False: There is a difference in the free energy of the overall reaction (energy of reactants - energy of products) between the catalyzed and uncatalyzed reaction
False - there is NO difference in the free energy
A ____ ⇆ T* (high-energy intermediate) ⇆ B (product)
A (substrate)
A reaction occurs if molecules contain enough ___ to overcome the energy
barrier to achieve T*
energy
Reaction _____ is determined by # of molecules that have sufficient
energy to overcome the barrier
rate or velocity
True or False: If free energy of activation is low: less molecules have enough energy to reach T* and reaction proceeds more slowly
False - If free energy of activation is low: MORE molecules have enough energy to reach T* and reaction proceeds more quickly
Why do enzymatic reactions have a more rapid reaction rate?
1) Enzyme provide an alternate reaction pathway
2) lower free energy of activation
3) more molecules possess enough energy to overcome barrier
An enzyme’s ____ is a complex molecular machine that facilitates
conversion of substrate into product
active site
Substrate binds and initiates conversion into ___
Substrate binds and initiates conversion into T*
What do amino acid residues at the active site do?
- Donate or accept protons
- Hydrogen bonding to promote T state formation
True or False: Enzymes are usually least responsive to changes in [S]
False - Enzymes are usually most responsive to changes in [S]
_____: # of substrate molecules converted to product per unit of time when enzyme levels are constant
Rate or velocity (V) of enzymatic reactions
What three factors influence enzymatic reaction velocity?
- substrate concentration [S]
- Temperature
- pH
With constant [E], reaction rate increases with ↑[S] until reaching ___
Vmax (max velocity)
High [S] eventually leads to ____ of all binding sites on enzymes, which causes
______ to level off
saturation; reaction rate
True or False: Once Vmax is reached, reaction rate increases with addition
of more substrate
Once Vmax is reached, reaction rate does NOT increase with addition
of more substrate
True or False: Increasing temperature generally increases the rate of a reaction
True
What happens to enzymes at extremely high or low pH?
Enzyme denatured
and no longer functional
An abnormal pH affects functionality of amino acids located in the active site and lowers what variable?
Vmax
Michaelis-Menten kinetics
Enzymes following Michaelis-Menten Kinetics show a ____ curve. Allosteric enzymes show a ___ curve
hyperbolic; sigmoid
___: describes how reaction velocity varies with [S]
Michaelis-Menten Kinetics
True or False: Enzyme (E) reversible combines with substrate (S) to form an enzyme-
substrate (ES) complex, which yields a product.
Free energy is generated.
What assumption does MM-Kinetics make?
1) [S]»_space; [E]
2) rate of ES formation = rate of ES breakdown
MM Equation?
Km =
[S] at ½ Vmax
Km demonstrates the affinity of enzyme for _____
substrate
Small Km means ___ affinity of enzyme for substrate and ___ [S] needed to reach Vmax
high; low
Large Km means ___ affinity of enzyme for substrate and __ [S] needed to reach Vmax
low; high
For Lineweaver burk plot:
If an inhibitor is covalently bound to enzyme, preventing it from interacting with S and resulting in “loss” of enzyme activity (i.e., lead poisoning), it is likely to be what type of inhibitor?
Irreversible Inhibitor
True or False: Reversible inhibitors use non-covalent binding with enzyme, which allows for recovery of enzyme
True
What are the two types of reversible inhibitors?
-Competitive
-Noncompetitive
Why are competitive inhibitors called “competitive”
They bind to SAME site as substrate – in that way, then, they compete with S to bind enzyme’s active site
How does a competitive inhibitor affect…
-Km apparent?
-Vmax?
Vmax = unchanged
Km apparent = increased
True or False: Statin drugs (like: Pravastin) are examples of non-competitive inhibitors?
False - Statin drugs (like: Pravastin) are examples of competitive inhibitors
Statin drugs inhibit rate-limiting step in ______
(HMG CoA => Mevalonic Acid)
cholesterol biosynthesis
Statin drugs compete with substrate HMG-CoA for
binding to _____
HMG CoA-reductase
What type of inhibitor bind to different sites than the substrate and prevents an enzymatic reaction from occurring?
Non-competitive inhibitor
How is it possible that non-competitive inhibitors can bind to either: a free enzyme or the ES complex?
The inhibitor binds to a different place than the substrate
How do non-competitive inhibitors affect….
-Km?
-Apparent Vmax?
Km = unchanged
Apparent Vmax = decreased
True or False: For non-competitive inhibitors, large quantities of substrate CAN overcome
inhibition
False - for non-competitive inhibitors, even large quantities of substrate cannot overcome inhibition
What type of inhibitors are Penicillin and Amoxicillin?
Mechanism of action?
Irreversible Inhibitors
Inhibition of bacterial cell wall synthesis
What type of inhibitors are ACE inhibitors (captopril, enalapril, and lisinopril)?
Reversible Competitive Inhibitors
NSAIDs are all ____inhibitors. However: acetylsalicylic acid (aspirin) is ____while ibuprofen is _____
competitive
irreversible
reversible
True or False: Increase [S] increases reaction rate/velocity
True
Other mechanisms (3) involved in regulating enzyme activity?
1) Allosteric regulation
2) Covalent modification
3) Induction/repression of enzyme synthesis
True or False: Allosteric enzymes usually contain only one subunit and catalyze the last step in a pathway
False - allosteric enzymes
often contain multiple subunits and frequently catalyze the committed, rate-limiting step in a pathway
True or False: Allosteric enzymes do not follow M-M kinetics
True (makes a sigmoidal curve)
What type of molecules regulate allosteric enzymes?
Effector molecules
_____: bind noncovalently at a site other than active site
Effector molecules
What type of effector reduces or inhibits enzyme activity?
What type of effector stimulates or increases enzyme activity?
Negative effector
Positive effector
What can effectors modify?
1) Vmax
2) Affinity of enzyme for S (K0.5)
3) or both
Homotropic effector are usually ___ effectors
Positive
Binding of substrate at allosteric site enhances activity at substrate-
binding site. This is known as ____
cooperativity
For a homotropic effector, what type of curve is visible? Why?
Sigmoidal curve
1) higher [S] initially
needed for reaction to proceed
2) substrate must bind at two different sites
True or False: In homotropic effectors, substrate = effector
True
True or False: In a heterotropic effector, substrate does not equal effector
True
____: downstream product in pathway
serves as effector for an enzyme required in an upstream reaction
Feedback Inhibition
An example of covalent modifications is: phosphorylation of specific ___, ___, or ____ residues:
Ser, Thr or Tyr
True or False: Phosphorylation only inhibits its target
False - phosphorylation may activate or inhibit its target enzyme
What are two ways to induce/repress enzyme synthesis?
1) Change rate of synthesis or degradation of enzyme
2) Slow mode of regulation (hours to days)
True or False: Allosteric and covalent modifications are slower
False - they are more rapid!(seconds to minutes)
