Lab 6: Reversible Inhibition Flashcards

1
Q

What type of inhibition was studied using what?

A

Reversible inhibition was studied using partially purified B-galactosidase

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2
Q

How was the degree of inhibition measured?

A

By the amount of synthetic substrate ONPG that produces ONP (a yellow colour) when cleaved

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3
Q

What determines first order?

A

[So] is much smaller than Km

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4
Q

What determines if a reaction is zero order?

A

If [So] is much larger than Km, v approaches Vmax

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5
Q

What is Km?

A

The substrate concentration when the reaction velocity is half its maximum velocity

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6
Q

Why are linear transformations often used instead of the hyperbolic function?

A

Because of errors associated with Vmax and Km with a hyperbolic plot

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7
Q

What do the X and Y intercepts, as well as the slope, show on a Lineweaver-Burk graph?

A

The slope shows Km/Vmax
The X-axis is for -1/Km
The Y-axis is for 1/Vmax

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8
Q

What type of inhibitor was calcium?

A

Mixed-type inhibitor

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9
Q

What type of inhibitor was sucrose?

A

Uncompetitive

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10
Q

What type of inhibitor was IPTG?

A

Competitive

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11
Q

What is Ki?

A

Dissociation constant for inhibitor binding to free enzyme

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12
Q

What is Kies?

A

Dissociation constant for inhibitor binding to enzyme-substrate complex, Kies

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13
Q

What are Kmapp and Vmaxapp?

A

the “apparent” values of Km and Vmax that are acutally observed in the presence of inhibitor for the Michaelis-Menten equation for inhibited enzymes

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14
Q

What makes a inhibitor competitive?

A

Binds at the active site and competes with substrate for enzyme

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15
Q

How do Vmax and Km change for competitive inhibition?

A

Vmax is unchanged, Km increases

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16
Q

What makes a inhibitor mixed-type?

A

Inhibit enzyme activity by combining both enzyme and enzyme-substrate complex

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17
Q

How do Vmax and Km change for mixed-type?

A

Vmax decreases, Km can increase or decrease

18
Q

What makes a uncompetitive inhibitor?

A

Binds only with the enzyme-substrate complex. Limiting case of mixed-type inhibition where Ki approaches infinity

19
Q

How do Vmax and Km change for uncompetitive?

A

Vmax decreases, Km decreases

20
Q

What makes a non-competitive inhibitor?

A

Special case of mixed type inhibition where Ki=Kies and Vmax decreases by the inhibition factor while Km is unchanged

21
Q

What is the equation for Km? What is it called?

A

The Michaelis- Menten Constant

Km= (K-1 + K2)/K1

22
Q

What did we change for the reaction? What was kept constant?

A

Inhibitor and Substrate concentrations were changed and the concentration of the enzyme was kept constant

23
Q

What were the reaction velocities measured for?

A
  • for varying [S] with fixed [I]

- for varying [I] with fixed [S]

24
Q

What was the enzyme used?

A

B-galactosidase

25
What is the substrate that is used?
ONPG
26
What are the inhibitors used?
2 mg/mLIPTG, 200 mg/mL calcium, 1 M sucrose
27
For the initial dilution of B-gal what is the rate of change needed for ONPG?
0.2 to 0.3 A405nm/min
28
Why do you need to keep enzyme on ice?
to minimize degradation
29
What do we use to blank the sample to read the velocities?
- B-gal assay buffer - substrate - diluted B-galactosidase enzyme
30
What are the units for velocity used in the lab?
delta A405nm/minute read at 15 second intervals for 2 minutes
31
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
32
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
33
What can be found using the initial velocity vs [S] graph?
Vmax and Km (just an estimate)
34
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
35
Which types of inhibitors have Ki values?
Competitive and mixed-type (Ki is for binding to the free enzyme)
36
Which type of inhibitors have Kies values?
Uncompetitive and mixed type (Kies is binding to the enzyme-substrate complex)
37
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)
38
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
39
What does the inhibitor bind for competitive inhibition?
Free enzyme at active site
40
What does the inhibitor bind for uncompetitive inhibition?
Enzyme-substrate complex NOT at active site
41
What does the inhibitor bind for mixed-type inhibition?
Free enzyme NOT at active site AND Enzyme-substrate complex NOT at active site
42
What does the Michaelis-Menten equation show?
The behavior of most enzymes but NOT the mechanism