Enzyme Kinetics Flashcards
What assumptions are made about substrate concentration when discussing Michaelis-Menten kinetics?
- The rapid equilibrium approximation: K1 and K-1 are small compared to Kp
- Initial rate/High [S]: [S] represents the free substrate concentration and is assumed to be close to the total substrate concentration because the reaction just began
- Initial Rate: The [ES] rapidly became steady because substrate is unlimited and [EP] is tiny
What is Vmax?
Vmax is the maximum velocity that would occur when all the enzyme is part of the enzyme-substrate complex
What is kp?
The reaction constant between the enzyme-substrate complex and the enzyme + product step
What is k-1?
Dissociation constant between the enzyme-substrate complex step and the enzyme + substrate step
What is the steady state assumption?
The concentrations of the intermediates of a reaction remain the same even when the concentrations of starting materials and products are changing
What does a high Kcat value signify?
Faster product production
What is the specificity constant?
Kcat/Km
Why is Kcat/Km the specificity constant?
The equation is basically the maximum product production per enzyme over the inverse efficiency of substrate binding.
What is a Lineweaver-Burk plot?
The plotting of Michaelis Menten kinetics as a double reciprocal
What can you tell by a Lineweaver-Burk plot?
- The slope is Km/Vmax
- y-intercept is 1/Vmax
- x-intercept is -1/Km
Why are inhibitors necessary in biological systems?
They regulate proteins
What is competitive inhibitor?
A type of reversible inhibition. The inhibitor competes with substrate for binding, then binds to the active site but does not affect catalysis.
What is a mixed inhibitor?
Inhibitor binds enzyme or enzyme-substrate complex. Binds to the regulatory site. Inhibits catalytic function and substrate binding.
What is the Michaelis constant? What does it represent?
Km represents a dynamic, in-progress reaction where the concentration of intermediate complexes does not change
How do you set up Michaelis-Menten conditions?
The concentration of substrate must be 100x larger than Ks and the fastest initial rate of product formation/substrate disappearance is measured
If given a graph with Velocity on the y-axis and [S] on the x-axis, how do you find Km?
Just like measuring the Kd, the Km is the x-coordinate at the 1/2 Vmax
What does Km approximate?
Km is a measure of roughly how much substrate is needed for full speed. The inverse substrate binding efficiency and the cellular [S]
What would happen is [S] was very different from Km?
The enzyme would be insensitive to changes
In regards to the Km value, what would indicate an inhibitor?
If the cellular levels of substrate is larger than the Km
In regards to the Km value, what would indicate an activator?
If the Km was larger than the cellular levels of substrate
Why are small Km values important?
The smaller the Km, the less E and S needed to make ES. Small Km values make the most efficient enzymes.
What is turnover?
The enzyme’s effect of turning substrate into product
What is the relation between turnover and Kcat?
They are the same. It’s the “limiting” rate of an enzyme catalyzed reaction
What is the Kcat equation?
Kcat=Vmax/[total E]
How is Vmax affected by competitive inhibition?
It stays the same
How does competitive inhibition affect Km?
Appears to increase
How would competitive inhibition appear on a Lineweaver-Burk plot?
The slope of the line would increase making the line more vertical because the Km increases and the Vmax stays the same (remember slope is Km/Vmax)
How is Vmax affected by uncompetitive inhibition?
Vmax decreases
How does uncompetitive inhibition affect Km?
Km appears to decrease
How would uncompetitive inhibition appear on a Lineweaver-Burk plot?
Like a normal because slope does not appear to change, the lines are parallel to the line of no inhibitor
What is an uncompetitive inhibitor?
The inhibitor binds to the Enzyme-Substrate complex. It does not affect substrate binding and inhibits catalytic function.
How is Vmax affected by mixed inhibition?
Vmax decreases
How does mixed inhibition affect Km?
Km appears to increase
How would mixed inhibition appear on a Lineweaver-Burk plot?
The slope increases, making a more vertical line
What type of inhibitions decrease the Vmax?
Uncompetitive and Mixed
What type of inhibitors decrease the Km?
Uncompetitive
What type of inhibitors increase the Km?
Competitive and mixed
What inhibitor keeps the same Vmax?
Competitive
What inhibitor keeps the Vmax and increases the Km?
Competitive
What inhibitor decreases the Vmax and decreases the Km?
Uncompetitive
What inhibitor decreases the Vmax and increases the Km?
Mixed
What is irreversible inhibition?
Something modifies the enzyme
How do irreversible inhibitors typically function?
By covalently modifying the enzme or enzyme-substrate complex by highly reactive groups. Others bind so tightly that there is no measurable release under physiological conditions
What can reveal Irreversibility?
- Fresh Enzyme and re-measuring kinetics
- Dilution of removal of inhibitor does not restore activity
What are suicide Inhibitors?
A subset of irreversible inhibitors
