13 - Enzyme Kinetics II Flashcards
How is product change over time usually measured?
Through absorbance
What is an example of measuring product change?
Beta-galactosidase: cleave into galactose and a red absorbance molecule
What is the Michaelis Menten curve?
v0 vs S
For increasing E, how does vmax change?
Vmax is proportional to E (vmax = kcatE)
For increasing E, how does Km change?
Km is independent of E
How come Km and kcat do not change with more E or S?
They are inherent properties of the enzyme
What properties does Km describe?
Relative binding affinity, conversion from S –> P
What does a low Km mean?
High affinity
What is the Lineweaver-Burk plot?
A linear form of the Michaelis Menten equation
What is the equation for the Lineweaver-Burk plot?
1/v0 = Km/(vmax*S) + 1/vmax
What is plotted in a Lineweaver-Burk plot?
1/v0 vs 1/S
What is the slope of a Lineweaver-Burk plot?
Km/vmax
What is the y-intercept of a Lineweaver-Burk plot?
1/vmax
What is the problem with the Lineweaver-Burk plot?
It can be inaccurate for low S (reciprocal)
What is the Hanes-Woolf plot?
Multiply the Lineweaver-Burke plot by [S] to remove reciprocal
What is inhibition?
Prevention of catalytic reaction (slow or halt)
What are the two classes of inhibition?
Reversible and irreversible
What is an example of an irreversible inhibitor?
Asprin
How does asprin work?
It transfers a group to the active site to prevent activity
What is an example of a reversible inhibitor?
Ibuprofin
How does ibuprofin work?
It is at equilibrium between the enzyme (bound and unbound)
Which type of inhibition is modifying the enzyme?
Irreversible
What time scale is needed for irreversible inhibition?
Life time of enzyme
What type of inhibition is a Kd process?
Reversible
What time scale is needed for reversible inhibition?
Therapeutic window (peaks)
What is Ki?
Dissociation constant for inhibitors to free enzyme
What are the three types of reversible inhibitors?
Competitive, noncompetitive, and uncompetative
What is a competitive inhibitor?
Bind to free enzyme
What is an uncompetitive inhibitor?
Bind to ES complex (or subsequent species, ES –> E + P)
What is a noncompetitive inhibitor?
Interferes with both processes (mixed) (E and ES)
What happens during competitive inhibition?
I resembles S but cannot undergo reaction, so I and S compete with E (decreases ES)
What happens at very high S for competitive inhibition and why?
The reaction will reach vmax, because it will outcompete
What kinetic parameters change with competitive inhibition?
Km
What kinetic parameters do not change in competitive inhibition?
vmax
What happens when I increases for a competitive inhibitor?
Apparent Km increases
What does a competitive inhibitor look like in a Lineweaver-Burk plot?
At increasing inhibitors, the lines will intersect at the y-axis (higher slopes)
How can you determine Ki for a competitive inhibitor?
Plot Km(app) vs. I, and the x-intercept is -Ki
What happens during uncompetitive inhibition?
S and I bind at the same time to different sites of the enzyme, affecting catalytic function (not substrate binding)
What are some mechanisms of uncompetitive inhibition?
May cause structural distortion in active site, making enzyme less competent, or may prevent product disassociation
What kinetic parameters change with an uncompetitive inhibitor?
vmax, Km
What kinetic parameters do not change with uncompetitive inhibitor?
vmax/Km (ratio)
What does an uncompetitive inhibitor look like in a Lineweaver-Burk plot?
At increasing I, there will be parallel lines (larger y intercept)
How can you determine Ki from an uncompetitive inhibitor?
Plot S/vo vs. I at increasing S levels, and they will intersect at -Ki’
What does Ki’ represent?
Dissociation constant of I from the ESI complex
What type of inhibition can allosteric be?
Uncompetitive or noncompetitive
How can the mechanism of inhibition be determined?
Through kinetic assays
What happens during noncompetitive inhibition?
I binds whether or not the substrate is bound (not active site) (interferes with both processes)
What kinetic parameters change with noncompetitive inhibition?
vmax, Km
What kinetic parameters do not change in noncompetitive inhibition?
None
What does noncompetitive inhibition look like in a Lineweaver plot?
At increasing I, the lines will intersect left of the y-axis (increasing slopes)
How can Ki and Ki’ be determined for a noncompetitive inhibitor if Ki = Ki’?
Plot S/vo vs I at increasing S, and they will intersect at -Ki
How can Ki and Ki’ be determined for a noncompetitive inhibitor if Ki does not equal Ki’?
Need two plots (1/vo vs. I and S/vo vs. I at increasing S levels) to determine parameters (where lines intersect)
What is the problem with Ki?
It is a lot of work to calculate Ki for every inhibitor
What is the IC50%?
The I that gives 50% inhibition at a specific enzyme and substrate concentration
What is the advantage of the IC50%?
It is quick and easy to calculate
If E and S are kept constant, what is IC50% a measure of?
Relative ability to inhibit enzyme
How can IC50% be calculated?
Plot vi/vo vs log(I), and find the I value where vi/vo = 0.5
What is the relationship between Ki and IC50%?
If Km and type of inhibition are known, the Cheng and Prusoff relationships can be used to calculate Ki from IC50%
What is the goal in drug discovery?
Want high affinity
What is the problem with high affinity inhibitors?
Difficult to ensure E «_space;I
What happens to IC50% if the affinity is very high?
The potency can be underestimated
When does problems with IC50% occur?
When Ki is close or less to [E]
How come [E] cannot be dropped too much?
Need to detect the reaction with a certain level of E
What is critical for tight binding inhibition?
The ratio of apparent Ki/Etot
What happens if Ki/Etot > 10?
Not tight binding, IC50% = Ki apparent
What happens if Ki/Etot is between 10 and 10^-2?
IC50% = Ki(app) + 0.5Etot
When happens if Ki/Etot < 0.01?
Dissociation is negligible, and Ki(app) cannot be determined (IC50% = 0.5Etot)
What is slow onset inhibition?
The binding of inhibitor to enzyme converts into a final complex
What is the formula for slow onset inhibition?
E + I –> EI –> EI*
What is the slow step for slow onset inhibition?
Structural reorganization (low koff, EI –> EI*)
What is koff for slow onset inhibition?
How rapidly the enzyme-inhibitor complex dissasociates
How some slow onset inhibitors are good drugs?
They dissociate slowly from the target enzyme, enzyme remains inhibited when drug concentration decreases, and have long residence time (slow dissociation)
