Lab 6: Reversible Inhibition Flashcards
What type of inhibition was studied using what?
Reversible inhibition was studied using partially purified B-galactosidase
How was the degree of inhibition measured?
By the amount of synthetic substrate ONPG that produces ONP (a yellow colour) when cleaved
What determines first order?
[So] is much smaller than Km
What determines if a reaction is zero order?
If [So] is much larger than Km, v approaches Vmax
What is Km?
The substrate concentration when the reaction velocity is half its maximum velocity
Why are linear transformations often used instead of the hyperbolic function?
Because of errors associated with Vmax and Km with a hyperbolic plot
What do the X and Y intercepts, as well as the slope, show on a Lineweaver-Burk graph?
The slope shows Km/Vmax
The X-axis is for -1/Km
The Y-axis is for 1/Vmax
What type of inhibitor was calcium?
Mixed-type inhibitor
What type of inhibitor was sucrose?
Uncompetitive
What type of inhibitor was IPTG?
Competitive
What is Ki?
Dissociation constant for inhibitor binding to free enzyme
What is Kies?
Dissociation constant for inhibitor binding to enzyme-substrate complex, Kies
What are Kmapp and Vmaxapp?
the “apparent” values of Km and Vmax that are acutally observed in the presence of inhibitor for the Michaelis-Menten equation for inhibited enzymes
What makes a inhibitor competitive?
Binds at the active site and competes with substrate for enzyme
How do Vmax and Km change for competitive inhibition?
Vmax is unchanged, Km increases
What makes a inhibitor mixed-type?
Inhibit enzyme activity by combining both enzyme and enzyme-substrate complex
How do Vmax and Km change for mixed-type?
Vmax decreases, Km can increase or decrease
What makes a uncompetitive inhibitor?
Binds only with the enzyme-substrate complex. Limiting case of mixed-type inhibition where Ki approaches infinity
How do Vmax and Km change for uncompetitive?
Vmax decreases, Km decreases
What makes a non-competitive inhibitor?
Special case of mixed type inhibition where Ki=Kies and Vmax decreases by the inhibition factor while Km is unchanged
What is the equation for Km? What is it called?
The Michaelis- Menten Constant
Km= (K-1 + K2)/K1
What did we change for the reaction? What was kept constant?
Inhibitor and Substrate concentrations were changed and the concentration of the enzyme was kept constant
What were the reaction velocities measured for?
- for varying [S] with fixed [I]
- for varying [I] with fixed [S]
What was the enzyme used?
B-galactosidase
What is the substrate that is used?
ONPG
What are the inhibitors used?
2 mg/mLIPTG, 200 mg/mL calcium, 1 M sucrose
For the initial dilution of B-gal what is the rate of change needed for ONPG?
0.2 to 0.3 A405nm/min
Why do you need to keep enzyme on ice?
to minimize degradation
What do we use to blank the sample to read the velocities?
- B-gal assay buffer
- substrate
- diluted B-galactosidase enzyme
What are the units for velocity used in the lab?
delta A405nm/minute read at 15 second intervals for 2 minutes
What type of graph is Vo vs. [S]? Why?
- hyperbolic
- the substrate is able to interact maximally with the free enzyme. As the substrate-enzyme complex reaches equilibrium the reaction will slow down. At saturation point then it plateaus
What happens at low [S] vs high [S] for the hyperbolic curve?
- low [S]: most of enzyme is in free E form. Rate of the reaction is proportional to [S] because the equilibrium of the reaction is pushed towards the formation of [ES] as [S] increases
- high [S]: most of the enzyme is in the ES form. The enzyme essentially becomes saturated. Vmax is reached because increasing [S] has little effect on the rate of the reaction. The reaction rate plateaus
What can be found using the initial velocity vs [S] graph?
Vmax and Km (just an estimate)
What can be found on the Dixon Plot? What are the axis?
1/Vo (Y) vs [I] (X axis) for each fixed substrate
Find the -Ki value (the x-axis where the lines intersect) which converts to a +Ki value
Which types of inhibitors have Ki values?
Competitive and mixed-type (Ki is for binding to the free enzyme)
Which type of inhibitors have Kies values?
Uncompetitive and mixed type (Kies is binding to the enzyme-substrate complex)
What can be found on the [S]/Vo versus [I] graph?
- made for the fixed [S]
- Kies
- -Kies is the x-axis where the lines intersect (give + value)
What makes competitive inhibition inherently different from uncompetitive and mixed-type inhibition?
- inhibitor binds at the active site
- means Vmax is unchanged because increasing the [S] can overcome the inhibition
What does the inhibitor bind for competitive inhibition?
Free enzyme at active site
What does the inhibitor bind for uncompetitive inhibition?
Enzyme-substrate complex NOT at active site
What does the inhibitor bind for mixed-type inhibition?
Free enzyme NOT at active site AND Enzyme-substrate complex NOT at active site
What does the Michaelis-Menten equation show?
The behavior of most enzymes but NOT the mechanism
