02c: Enzyme Kinetics and Inhibition Flashcards
In unimolecular irreversible reaction, rate of P formation equals (X) and is directly proportional to (Y).
X = rate of A disappearance
Y = [A]
First order rate constant units.
1/s
Second order reaction rate constant units.
1/Ms
In unimolecular reversible reaction: large Keq favors (reactants/products).
Products
What’s the plot shape of substrate concentration versus reaction velocity in typical enzyme-catalyzed reaction?
Hyperbolic
Zero order reaction is found at what substrate concentration? The reaction velocity equals:
When [S] much greater (20x) Km
Vo = Vm
List Michaelis-Menten assumptions.
- ES forms
- No back reaction
- Initial v used
- [ES] in steady state
- Negligible substrate depletion
How does a larger Km affect the plot/curve shape?
Flattens out curve
Kd is known as (X) and tells you about (Y).
X = dissociation constant Y = binding affinity
Small Km is indicative of (strong/weak) binding.
Strong
Kcat is formally termed:
Turnover number
Catalytic efficiency of enzyme represented by:
Kcat/Km
What defines a “perfect enzyme”?
Kcat/Km in range of
10(^8) - 10(^9)
Units for catalytic efficiency:
1/Ms
It’s recommended to use (linear/nonlinear) regression of data to determine Km and Vm.
Nonlinear (hyperbolic)
Is it possible to linearize Michaelis-Menten?
Yes - take reciprocal of both sides to get Lineweaver-Burk plot
Lineweaver-Burk plot slope represents:
Km/Vm
Lineweaver-Burk plot x-intercept represents:
-1/Km
Lineweaver-Burk plot y-intercept represents:
1/Vm
Increasing Vm will have what effect on Lineweaver-Burk plot?
Bring y-intercept closer to origin
Increasing Km will have what effect on Lineweaver-Burk plot?
Bring X-intercept closer to origin
A “perfect enzyme” will have what difference in Lineweaver-Burk plot compared to another enzyme?
Smaller slope
Can Kcat be determined from (michaelis-menten/lineweaver-Burke) plot? What’s the equation?
Either, as long as [E]t and Vmax are determined;
Vm = Kcat*[E]t
Bi-substrate reaction classes:
- Sequential
2. Ping-pong
All substrates bind in specific order before any product released describes which bi-substrate reaction class?
Sequential (ordered)
All substrates bind randomly before any product released describes which bi-substrate reaction class?
Sequential (random)
One substrate binds and releases product before second substrate binds describes which bi-substrate reaction class?
Ping-pong (double-displacement)
(Competitive/uncompetitive/non competitive) inhibitors are irreversible.
None - all are reversible
Competitive inhibitors bind to:
Free enzyme only
T/F: competitive inhibitors don’t affect the amount of ES complex formed.
False
How does Km change in presence of competitive inhibitor.
It doesn’t! Only apparent value changes
How does apparent Km value change in presence of competitive inhibitor?
Increase
How does apparent Vm value change in presence of competitive inhibitor?
No change
How does apparent catalytic activity value change in presence of competitive inhibitor?
Decrease
Uncompetitive inhibitors bind to:
ES complex only (not at active site)
How does uncompetitive inhibitor hinder reaction?
Distorts active site, making it catalytically inactive
How does apparent Vm value change in presence of uncompetitive inhibitor?
Decreases
How does apparent Km value change in presence of uncompetitive inhibitor?
Decreases
How does apparent catalytic activity value change in presence of uncompetitive inhibitor?
No change
At low [S], which inhibitor(s) decrease rate?
Competitive and Noncompetitive
At low [S], which inhibitor(s) don’t change rate?
Uncompetitive
Noncompetitive inhibitors bind to:
Free enzyme or ES complex
Do (X) inhibitors bind with greater Ki to free enzyme or ES complex?
X = non-competitive
Bind with same Ki to either
How does noncompetitive inhibitor hinder reaction?
Distorts enzyme structure, preventing alignment of catalytic center
How does apparent Vm value change in presence of noncompetitive inhibitor?
Decreases
How does apparent Km value change in presence of noncompetitive inhibitor?
No change
How does apparent catalytic activity value change in presence of noncompetitive inhibitor?
Decreases
Enzymatic activity can be precisely regulated in which ways?
- Allosteric control
- Regulatory proteins
- Reversible covalent modifications
- Proteolytic activity
Allosteric enzymes do not obey (X) because the [S] v. Vo plot has which shape?
X = michaelis-menten kinetics
Sigmoidal shape
Sigmoidal curves result from (X) cooperativity, indicating which change in Km?
X = positive
Decrease Km with increase in [S]
In allosteric control, (X) effector is same as substrate and (Y) effector is different from substrate.
X = homotropic Y = heterotropic
Does allosteric control change Km or Vm?
Can change either or both
Are allosteric enzymes monomeric or oligomeric?
Can be either
ATCase is an enzyme that catalyzes:
first step in pyrimidine biosynthesis pathway
(X) is a negative effector of the ATCase enzyme.
X = CTP
Describe structure of ATCase.
6 catalytic subunits (trimers) and 6 regulatory subunits (dimers)
ATCase is in equilibrium between which two states? Which is favored?
Tense and relaxed;
Tense favored (200:1)
(X), the ATCase substrate, stabilizes which of the two enzyme states?
X = aspartate
Relaxed state
The final product of ATCase pathway, (X), stabilizes which of the two enzyme states?
X = CTP
Tense state
Name a positive effector of ATCase.
ATP
The positive/negative effectors of ATCase bind which subunits on enzyme?
Regulatory
Give example of reversible, covalent modifications of enzymes.
Phosphorylation and de-phosphorylation
T/F: Phosphorylation always increases enzyme activity.
False
Kinases (add/remove) (X) groups to/from proteins using:
Add
X = phosphate
ATP
Phosphatases (add/remove) (X) groups to/from proteins using:
Remove
X = phosphate
H2O (hydrolysis)
