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