Enzymes Flashcards
(105 cards)
1
Q
What are enzymes?
A
Biological catalysts. Usually proteins.
2
Q
What are some features of enzymes?
A
- Catalyse metabolic reactions
- One reaction = one enzyme
- Specific
- Lower activation energy
3
Q
What is the transition state?
A
The maximum value of energy, has to be reached for the reaction to happen
4
Q
What does the rate of reaction depend on?
A
How many reacting molecules acquire enough activation energy to reach and pass through the transition state
5
Q
Why can’t the body use pH or temperature as catalysts?
A
Can’t change these without harming the body but need to accelerate the reaction
6
Q
What is Rate Enhancement?
A
- Enzymes increase the rate, the reaction would still happen without it but a lot slower
- Enzymes determine the time scale for living organisms
7
Q
What does the rate of reaction depend on?
A
Size of the energy barrier and the reaction taken between S and P
8
Q
What does the substrate and product tell us?
A
The equilibrium of the system but not the kinetics - how fast S goes to P
9
Q
What is meant by equilibrium in the system?
A
Stable. Concentrations of reactants and products are constant, ratio does not vary. Forward and reverse reactions occur at equal rates
10
Q
What is the rate of reaction proportional to?
A
The concentration of reactants
- Rate A to B ∝ [A]
11
Q
What are K values?
A
Tell us how fast a reaction happens
12
Q
How do you calculate catalytic power?
A
ratio of catalysed/ uncatalysed rates
13
Q
What is the equation for equilibrium?
A
Keq = [B] / [A]
14
Q
What is a unimolecular reaction?
A
A → B
- If you increase conc of A the rate of reaction increase
- Linear relationship
15
Q
Why is the relationship between substrate conc and rate non linear?
A
Enzymes become limiting as there are no more available active sites
16
Q
What is Vmax?
A
Maximum rate that a given concentration of enzyme can achieve
17
Q
What is Km?
A
Substrate concentration giving a rate of Vmax/2
18
Q
What are the assumptions of Michaelis-Menten plot?
A
- Progress from the activated ES to product is irreversible
- Substrate is available in large amounts compared to enzyme
- ES complex is at a steadu state
19
Q
What are the two ways that ES can disappear?
A
- Dissociate back to E+S
- Form E+P
20
Q
How do you work out the rate of enzyme catalysed reaction?
A
V = [ES].k2
21
Q
Why can’t you measure [ES]?
A
Know how much E and S but as soon as the reaction starts ES forms too.
22
Q
What is the equation for the total amount of enzymes?
A
Etot = [E] + [ES]
23
Q
What is the Michaelis Menten equation?
A
V= Vmax[S]/ Km + [S]
24
Q
Why is there a mximum rate?
A
- Doesn’t have enough enzyme capacity
- At a fixed conc they become limiting
25
What represents the forward reaction?
K1
26
What represents the backwards reaction?
K-1
27
Describe Km.
- Related to rate
- Rate constancts that lead to breakdown of ES
- Km is dependent on ES concentration
28
What happens if Km is high?
Es is falling apart = unstable. Weak binding, low affinity
29
What happens if Km is low?
Stable = forming ES. Strong binding, high affinity.
30
What is the equation of Km?
K-1 + K2/K1
31
How do we plot a straight line from a curved Michaelis Menten graph?
Use the reciprocal, Linewearver - Burk plot
32
What is the equation for the Lineweaver- Burk plot (1/V)?
Km + [S]/Vmax*[S]
33
What is the Lock and Key state?
- Specifically recognises
- Active site complementary to substrate
- Non-covalent bonds formed
- Rigid state doesn't really cover how product is formed as substrate needs to change shape
34
What is the Induced Fit state?
- Active site forms in response to substrate bind, mimics shape
- Puts strain on the enzyme and substrate
- Lowers transition state and stabilises it
- Compromises specificity
35
Why does the transition need to be reached?
For substrate to be turned into produced
36
What is the energy landscape of lock and key?
Vmax and Km both decrease
37
Why do Vmax and Km decrease in the lock and key energy landscape?
- Enzyme substrate binding favoured
- Energy goes down at ES
- At transition - goes up - doesnt explain any energy to drive this state
- Energy goes down at EP
38
What is the energy landscape of induced fit?
Vmax and Km are both increased
39
Why do Vmax and Km increase in the induced fit energy landscape?
- SLight lowering of energy but not much
- ES goes down
- Substrate higher than product
40
How is efficiency in an enzyme measured?
Substrate affinity, turnover number and substrate specificity
41
What does Km reflect?
The availability of substrates in biological systems
42
What does Vmax depend on?
Enzyme concentration (reveals turnover number)
43
What is the turnover number?
Number of substrate molecules converted into product by an enzyme in a unit of time when the enzyme is fully saturated with substrate
44
How do we measure intrinsic enzyme activity?
Kcat
45
What is Kcat?
Catalytic constant of the enzyme, rate of substrate turnover
46
What does Kcat measure?
Number of moles of product generate per mole of enzyme per second
47
What has the highest known turnover number?
Catalse
48
What does Kcat/Km work out?
'Enzyme eficiency'
49
Why do Kcat/Km ratios vary?
Some reactions are chemically harder to do than others
50
What happens to the efficiency if Km rises?
Efficiency decreases
51
What is an example of a very specific enzyme?
Endonucleases
52
Why do enzymes need to be regulated?
Not efficient to have all enzymes producing if you don't want lots of product
53
How can enzymes be regulated?
Through effectors (inhibitors)
54
What are the 2 types of inhibition?
1. Competitive
| 2. Non-competitive
55
What is competitive inhibition?
Substrate and inhibitor compete for the active side
56
What is non-competitive inhibition?
Binding to a distinct site (allosteric state). Causes an conformational change in the enzyme
57
What is an example of competitive inhibition?
Tamiflu - influenza drug
58
What is an example of non-competitive inhibition?
Ibuprofen - inhibits Cox2, blocks pain and inflammation
59
What happens to Km during competitive inhibition and why?
Km increases, needs more substrate to reach 1/2Vmax
60
What happens to Vmax during competitive inhibition and why?
Vmax will still be reach it will just take longer
61
What happens to Km during non-competitive inhibition and why?
Km doesn't change because you're not changing substrate concentration
62
What happens to Vmax during non-competitive inhibition and why?
Vmax will decrease because you'll never reach the maximum rate
63
What is ATCase used in?
First step in pyrimidine biosynthesis
64
What is ATCase regulated by?
Pathway product CTP
65
Why does there need to be the right amount of ATCase?
DNA will be wrong - too many mutations
66
What is the substrate of ATCase?
ASP
67
How does inhibition work in ATCase?
- CTP bind to non-catalytic site
- Re-organisation of Quaternary structure
- Active site is no longer formed
68
What is the structure of ATCase?
- Big enzyme, small active site
| - Substrate analogues bound by H bonds to active site alpha side chains
69
What is the evidence for distinct catalytic and regulatory sites on ATCase?
Treatment with organic mercury containing compounds that bind cysteine cause ATCase to dissociate
Separate based on size/charge
Contains distinct catalytic and regulatory subunits
Catalytic → retains ATCase but is not CTP sensitive
Regulatory → no enzyme activity but binds CTP
70
Describe the T state in ATCase?
T state = poor substrate binding. When CTP binds it stabilises the T state
71
Describe the R state in ATCase?
HIghly active, favours substrate. When CTP binds unable to transition to R state
72
What happens when CTP binds to ATCase?
Binding of CTP stops the move between T and R state. Stabilies T state. Can't bind.
73
What happens when ATP binds to ATCase?
Stabilises the R state
74
What shape does the ATCase graph have?
Sigmoidal shape
75
What type of enzyme is ATCase?
Cooperative enzymes
76
What occurs at the active site?
Catalysis
77
Describe the active site.
Relatively small compared to entire protein. 3D cleft. Formed from amino acid residues. Active site residues not near in the primary structure - folding. Rest of protein is the supporting structure.
78
What are the different mechanisms of the active site?
```
Covalent catalysis
Acid base catalysis
Metal ion catalysis
Proximity and orientation effects
Electrostatic catalysis
```
79
Describe the main features of Serine proteases.
- Large family of enzymes
- Hydrolyse proteins
- In digestive tract, blood clotting, immunity
80
How are serine proteases involved in digestion?
Inactive form that can be cut down to an active form eg trypsin.
81
How are serine proteases involved in blood clotting?
Factor 10
82
What is the active site of serine protease?
Catalytic triad. Includes a crucial serine
83
What does the serine do in serine proteases?
Acts as a nucleophile that attacks alpha carbon and hydrolyses. End up with a carboxylic and amine product
84
How are oxyanions formed in serine proteases?
Pulls Asp and His closer together forming an oxyanion. Pulls a proton from Ser making it active. (His acts as a base and Ser acts as an acid)
85
What is covalent catalysis?
Oxyanion transition state intermediate is formed by a covalent bond.
86
What is the role of the backbones amines of Ser195 and Gly193?
Stabilise the transition state in the oxyanion hole
87
What is the oxyanion hole?
Cavity in the active site where the oxyanion intermediate can fit.
88
What is the interaction with oxyanion hole known as?
Electrostativ catlysis. Lowers activation energy
89
Describe the process of the oxyanion
rally of electrons → peptide bond breaks → amine still interacting with the active site → ser covalently attached to carboxylic → H bond breaks → amine released → hydrolysis to break covalent bond
90
What happens when the substrate enters the oxyanion?
Asp → proton → His → oxyanion → transition state
91
Describe Chymotrypsin?
Favours substrates. Amino acid N terminal of the hydrolysis is bulky - usually an aromatic side chain.
92
How does Chymotrypsin work?
Cleaves peptides on the carbonyl side of aromatic residues.
93
Describe the active site pocket of Chymotrypsin?
Surrounded by hydrophobic residues. Large enough to accommodate aromatic residues
94
What does Trypsin do?
Cleaves peptides on the carbonyl side of basic amino acids.
95
Describe the active site of Trypsin.
Active site pocket: negatively charged residue
96
WHat does Elastase do?
Cleaves peptides on the carbonyl side of small neutral residues
97
Describe the active site of Elastase.
Shallow pocket with bulky residues at its opening
98
Compare Chymotrypsin and Factor X?
- 50% amino acids identity
- Same fold
- Same arrangement of active site residues
- Divergent evolution from common ancestor
- Different biological functions
99
What is meant by saying the three residues of the catalytic triad are high conserved?
Active sites had these 3 residues
100
What do some enzymes require to carry out catalysis?
Metal ions. Coenzymes.
101
What does carbonic anhydrase use?
Zinc
102
What roles are carbonic anhydrase involved in>
Gas exchange. Regulates pH. Control water balance
103
Where is the zinc found in carbonic anhydrase?
3 His hold a zinc
104
How does Zinc work in order to speed up catalysis?
- Zinc ion activates water, stabilises a hydroxide ion at neutral pH which can acts as a base
- Hydroxide bound form is part of the transition state
- Zinc stabilises this because its strongly acidic
- No covalent intermediate
105
How does carbonic anhydrase achieve rapid removal of the proton from the active site?
Fourth His residue shuffles the proton to the protein surface.
