Allosteric enzymes and Inhibitors Flashcards
1
Q
What is an inhibitor?
A
Any molecule that acts to reduce the rate of an enzymatic reaction
2
Q
What can inhibitors be composed of?
A
Small chemicals or larger polymers (including other protein)
3
Q
What can act as competitive inhibitor?
A
Drugs
4
Q
What does a good inhibitor do?
A
Increase the Km of an enzyme
5
Q
What substrate does sildenafil mimic?
A
Phosphodiesterase 5
6
Q
What is a competitive inhibitor?
A
Resembles the substrate so if can bind to the active site but doesn’t react like the actually substrate
7
Q
Can a competitive inhibitor undergo a reaction with the enzyme?
A
No
8
Q
Finish the sentence:
The enzyme-inhibitor complex, is catalytically…
A
inactive
9
Q
What does competitive inhibitors reduce?
A
The concentration of free enzyme available for substrate binding
10
Q
Can a higher concentration of substrate over come the effects of the competitive inhibitor?
A
Yes
11
Q
Why does the inhibitor make the Km look larger on a Michaelis-Menten?
A
As the binding of the inhibitor and the substate to the enzyme are mutually exclusive
12
Q
What is v equal in the Michaelis-menten equation?
A
V = Vmax x [S] / aKm + [S]
13
Q
What is the double reciprocal of V = Vmax x [S] / aKm + [S]?
A
1/v = (aKm/Vmax)1/[S] + 1/Vmax
14
Q
What remains constant with competitive inhibitor on a line weaver burn plot?
A
Vmax
15
Q
What is an uncompetitive inhibitor?
A
Inhibitor binds directly to the enzyme-substrate complex but not to the free enzyme. (active site)
16
Q
What does uncompetitive inhibitors rely on?
A
Inhibitor affects the catalytic function the enzyme not the its substrate binding
17
Q
What is Kis?
A
The dissociation constant and has the units M
18
Q
What is Ki?
A
The dissociation constant and has units M
19
Q
What is Ki equal to?
A
[E][I]/[EI]
20
Q
What is Kis equal to?
A
[ES][I]/[ESI]
21
Q
Finish the sentence:
The enzyme-substrate-inhitibor complete, is catalytically…
A
inactive
22
Q
When are uncompetitive inhibition significant in?
A
Multi-substrate enzymes
23
Q
What does a uncompetitive inhibition do to Vmax/Km?
A
Nothing remains constant
24
Q
What does uncompetitive inhibition do to Km and Vmax?
A
Decrease
25
Is Vmax reversed by increasing the concentration of substrate when uncompetitive inhibition is occurring?
Nothing - Cant to be reversed
26
What is a mixed inhibitor?
Binds to enzyme sites that participate in both substrate binding and cataslyse
27
What is non-competitive inhibition?
```
Ki = Kis
a = a'
```
28
Is a non-competitive inhibitor the same an uncompetitive inhibitor?
No
29
Why is non-competitive inhibitor the different to an uncompetitive inhibitor?
Decrease in Vmax
Constant Km
No-competitive is irreversible
30
What does irreversible inactivation resemble?
Non-competitive inhibition
31
What do you call an inhibitor which binds to irreversibly to an enzyme?
Inactivator
32
What do inactivators reduce?
Level of [E]T = Vmax without changing Km
33
What is similar between non-competitive and inactivators on the double reciprocal plot?
Lines intersect on the 1/[S] axis
34
How can amino acids be inactivators?
Specific amino acid residues which have be chemical modified by reagents
35
What effect does competitive inhibition have on Km?
Increase
36
What effect does uncompetitive inhibition have on Km?
Decrease
37
What effect does mixed inhibition have on Km?
Increase
38
What effect does non-competitive inhibition have on Km?
None
39
What effect does competitive inhibition have on Vmax?
None
40
What effect does uncompetitive inhibition have on Vmax?
Decreases
41
What effect does mixed inhibition have on Vmax?
Decreases
42
What effect does non-competitive inhibition have on Vmax?
Decreases
43
How can we identify what types of inhibitors are present?
Using Lineweaver-Burk plot
44
What are multi-subunit enzymes?
- Made from more than one protein chain
- Can be identical to different chains
- The binding of one chain can influence the binding to other subunits
- Leadings to co-operativity between the subunits
45
Give an example of multi-unit enzymes:
Aspartate transcarbamoylase (ATCase)
46
How can you regulate enzyme activity?
- Control of enzyme availability
| - Control of enzyme activity
47
How do you control enzyme availability?
- Amount depends on rate of synthesis and rate of degradation
- Rate controlled by cell and can change dramatically over time
48
How do you control enzyme activity?
Directly regulated by structural alterations which influence substrate binding affinity
-Allosteric mechanism
49
What is an active site?
The region where the binding of substrate and catalysis takes place
50
What is the average size of an enzyme?
300 amino acids
51
What do the rest of the proteins present in the enzyme which aren't involve in the active site do?
- overall structure
- Ensure correct shape
- Allow for regulation
52
What are allosteric effectors?
Any molecule that can bind to an enzyme away from the active site and change the enzyme's activity for its substrate
53
What do allosteric activators do?
Increase the rate of reaction
54
What do allosteric inhibitors do?
Reduce the rate of reaction
55
What molecules have inherent flexibility?
Proteins
56
What is inherent flexibility?
Small changes in the structure in one part of the protein can be communicated throughout the entire structure.
57
What can inherent flexibility affect?
The fit of the active site to substate and transition state
58
What does allostery refer to?
The situation where a chemical binds to an enzyme away from the active site and influence the kinetic properties of the enzyme
59
Where doe the catalysis take place with an enzyme and a substrate?
Active site
60
What is the first step in the biosynthesis of pyrimidines?
ATCase catalyses
61
What does carbamoyl donate in the pathways leading to arginine as well as pyrimidines?
Carbamoyl group
62
What does aspirate and carbamoyl phosphate produce which is only used in synthesis of pyrimidine nucleotides?
CTP -Cytidine triphosphate
63
What is the allosteric behaviour of ATCase?
Substates bind cooperatively to the enzyme
- Allosterically inhibited
- Allosterically activated
64
How is ATCase allosterically inhibited?
By Cytidine triphosphate
65
How is ATCase allosterically activated?
By adenosine triphosphate
66
What is the curve for ATCase?
Sigmoidal
67
What does CTP do to ATCase enzyme's catalytic rate?
Decreases
68
What does ATP do to ATCase enzyme's catalytic rate?
Increases
69
What are allosteric effecters called when comparing to competitive inhibitors?
K-system effectors
70
What is the difference of allosteric effecters to competitive inhibitors?
-Affect Km and not Vmax
71
How is ATCase regulated?
By ATP and CTP
72
What is the structure of ATCase?
- Subunit composition C6R6
- Arranged as two sets of trimers of the catalytic subunits
- 3 sets of regulatory dimers
- Each dimer joins two trimers
73
What is r in the ATCase structure?
Regulatory subunit
74
What is c in the ATCase structure?
Catalytic subunit
75
What type of inhibitor is PALA?
Competitive Inhibitor
76
Describe the active site of ATCase:
- Crystal strutcure
- Substate bound to subunit interface
- Site lying at boundaries between pairs of c chains within catalytic trimer
- Each trimmer contributes three active sites
77
What does the binding of PALA cause to an enzyme?
Major changes in the quaternary structure
78
What two distinct quaternary forms do ATCase have?
-Absence of substrate or substrate analogous
OR
-Substrates or substrate analogous are bound
79
What are the forms of ATCase referred to?
T state
| R state
80
What is R state?
Relaxed when substate bound
81
What is T state?
Tense when no substrate
82
How do you go from a T state to and R state transition?
- Cataltyic trimers separate along molecular three fold axis by 12A
- Rotate around three fold axis by 10 degrees
- The regulatory dimers rate clockwise around the two fold axis by 15 degree
- Separate by 4A along the three fold axis
83
Where do CTP and ATP bind to on ATCase?
Outer edge of regulatory subunit about 50A away from catalytic site
84
In which state (T OR R) does CTP bind to increase stability?
T-state
85
In which state (T OR R) does ATP bind to increase stability?
R-state
86
What happens when CTP binds to R-state?
Induces contraction in r dimer that cause c trimers to come together by 0.5A becoming less active
87
What happens when ATP binds to T-state?
Causes c trimers to move apart by 0.4 A becoming more active
88
Finish the sentence:
| The T state and R state are in....
equilibrium
89
What does the binding of CTP do to the equilibrium?
Shifts the equilibrium to T-State which decreases the net enzyme activity and reducing the rate of N-carbamoylaspartate generation
90
Do allosterically regulated enzymes follow the Michaelis-Menten kinetics?
No
91
What curve do allosterically regulated enzymes make?
Sigmoidal curve
92
Why do allosterically regulated enzymes make sigmoidal curve?
The biding of substrate to one active site favours the conversion of the entire enzyme in to R-State increasing the activity at the other active sites
93
What are the basis for sigmoidal curve?
Mixture of two Michaelis-Menten enzymes
- One with high Km (low affinity) for substrate (T-state)
- One with low Km (high affinity) for substrate (R-state)
94
Does a high Km have a low or high affinity for substrate?
Low
95
Does a low Km have a low or high affinity for substrate?
High
96
What is positive cooperatively?
Binding of substrate to one unit increases likelihood of other subunits binding substrate
97
What is negativity cooperatively?
Binding of substrate to one subunit to reduce the likelihood of other subunits binding substrate
98
Which is more likely negative or positive cooperatively?
Positive cooperatively
