enzymes Flashcards
1
Q
What is an Enzyme?
A
Globular protein
Biological catalyst that differs from a chemical catalyst
2
Q
enzymes: ribozymes
A
catalytic RNA molecules with no protein component
3
Q
enzymes are biological catalysts that
A
- Catalyses very high reaction rates
- Shows great reaction specificity
- Work in mild temperature/pH conditions
- Can be regulated
4
Q
Cofactor =
A
Non-protein component needed for activity
eg- ions
5
Q
cofactor in glucose-6-phosphate
A
Mg2+
6
Q
cofactor in pyruvate kinase
A
K+
7
Q
cofactor in catalase, peroxidase
A
Fe2+, Fe3+
8
Q
Coenzyme
A
Complex organic molecule, usually produced from a vitamin
9
Q
coenzyme from riboflavin
A
FAD
10
Q
coenzyme from Niacin
A
NAD+
11
Q
coenzyme from pantothenate
A
Coenzyme A
12
Q
Prosthetic group =
A
Cofactor covalently bound to the enzyme or very tightly associated with the enzyme
eg- haem in haemoglobin
13
Q
Apoenzyme =
A
The protein component of an enzyme that contains a cofactor
14
Q
Holoenzyme =
A
“whole enzyme” – the apoenzyme plus the cofactor(s)
15
Q
Substrate =
A
Molecule acted on by the enzyme
16
Q
Active site =
A
Part of the enzyme in which the substrate binds and is acted upon
17
Q
Oxidoreductases - type of reaction
A
Transfer e-
18
Q
Transferases - type of reaction
A
Group transfers
19
Q
Hydrolases - type of reaction
A
Hydrolysis (transfer chemical groups to water)
20
Q
Lyases - type of reaction
A
Form, or add groups to double bonds
21
Q
Isomerases - type of reaction
A
Transfer groups within molecules (form isomers)
22
Q
Ligases - type of reaction
A
Formation of C-C, C-S, C-O and C-N bonds (coupled to ATP cleavage)
23
Q
Enzymes do not
A
- Move reaction equilibria
- Make a non-spontaneous reaction spontaneous
24
Q
Enzymes do
A
- Increase rates of spontaneous reactions
- Lower the activation energy of biochemical reactions
- Accelerate movement towards reaction equilibrium
25
“Useful” energy generated from cellular reactions is termed
Gibbs Free-Energy (G), originally called “available energy”
26
Spontaneous reactions must have a
–ve ΔG value as they will decrease enthalpy (H) and/or increase entropy (S)
27
Spontaneous reaction isn’t
instantaneous because of the energy barrier
28
Energy barrier =
energy required to position chemical groups correctly, bond rearrangements, e- rearrangements, etc...
29
Transition state shows
the moment that chemical bonds are formed and broken. the top bit of curve.
30
Addition of an enzyme to a spontaneous reaction
lowers the activation energy
| Enzymes allow the reaction to proceed via different route
31
Enzymes form non-covalent bonds with
substrate molecules, called the “binding energy” allowing them to take the reaction through a different path of reaction intermediates
32
No enzyme =
high activation energy ( high delta G)
33
CATALYSIS =
Active site complementary to transition state
34
How do enzymes reduce activation energy?
- Entropy reduction
- Desolvation
- Induced fit
35
Explain Entropy reduction
- Molecules in free solution will only react by “bumping” into one another
- Enzymes “force” the substrate(s) to be correctly orientated by binding them in the formation they need to be in for the reaction to proceed
36
Explain Desolvation
Weak bonds between the substrate and enzyme essentially replace most or all of the H-bonds between substrate and aqueous solution
37
Explain Induced fit
Conformational changes occur in the protein structure when the substrate binds
38
If we changed the substrate concentration [S] we would change
the initial rate of a reaction.
More substrate = higher initial rate of reaction
- As the reaction proceeds, the substrate is used up and the rate of reaction changes
- Initial velocity (V0) can be studied if we assume that initial [S] does not change – this only really works if you have loads of S
39
When substrate concentration becomes so large that V0 changes are vanishingly small you get
maximum reaction velocity, Vmax.
| Vmax occurs because all of the enzyme active sites are saturated with substrate
40
Michaelis-Menten equation
| when you plot V0 against substrate concentration you get a
hyperbolic curve.
| formation of an enzyme-substrate complex (ES)
41
Michaelis-Menten equation
Model states that the first part of the reaction (to produce ES) occurs reversibly
Second part of the equation (to produce E and P) occurs more slowly than the first part - rate limiting step
42
Michaelis-Menten equation:
| If 2nd part is slower it must
limit the rate of the overall reaction, so the overall rate of reaction must be proportional to the amount of ES
- In other words, more ES would give a higher overall reaction rate and less would give a slower overall reaction rate
43
V0 usually equates to the
steady state of a reaction, so study of these initial rates of reaction is termed “steady state kinetics”
44
M-M equation was derived from their hypothesis that t
he rate-limiting step of an enzymatic reaction is the breakdown of the ES complex to give free enzyme and product
45
V0 =
initial reaction velocity
46
Vmax =
maximum reaction velocity
47
What’s the point of the M-M equation?
The equation accounts for the hyperbolic curve you see when a reaction proceeds
- At low [S] (i.e. the [S] is much less than Km) the M-M equation looks like,
V0 = Vmax[S]
Km
At high [S] ([S] is much greater than Km) the equation looks like,
V0 = Vmax
48
Km is equivalent to
the substrate concentration at which the initial reaction rate is half of the maximum reaction rate
49
The “better way” of experimentally defining the Vmax and the Km is to
draw a Lineweaver-Burk plot using the same data as was used to draw the M-M hyperbolic curve
50
Lineweaver-Burk equation makes a
straight line graph
51
Km = k-1 /k1
This can also be termed the dissociation constant, Kd of the ES complex
52
Km is the
ratio of rate constant for breakdown of ES to E + S compared to the rate constant for formation of ES from E + S
53
larger Km values indicate
a less stable ES complex
54
smaller Km values indicate
a more stable ES complex
55
Km gives you a clue to the
affinity of the enzyme with it’s substrate
56
Vmax tells you
how fast a reaction is proceeding when the enzyme is saturated with substrate
57
when [S] is equal to the Km, the M-M equation looks like,
V0 = Vmax
| 2
58
isozymes
are different proteins but they catalyse the same reaction
59
Glucokinase and hexokinase are
isozymes.
both catalyse
Glucose + ATP ---> Glucose-6-phosphate + ADP
however have different kinetics
60
When [blood glucose] goes UP after a meal,
the glucokinase activity increases but hexokinase activity does not respond as it is already working at its Vmax – this property allows glucokinase to respond proportionally depending on the [blood glucose]
61
When [blood glucose] is LOW,
gluconeogensis releases glucose from the liver, but glucokinase cannot catalyse glucose back into glucose-6 phosphate under these conditions allowing glucose to be used by the body
62
Glucokinase km(affinity for glucose) and Vmax are
high
63
hexokinase Km (affinity for glucose) and Vmax are
low
64
Enzymes in the wrong place
tell us about disease.
eg- increased plasma levels of intracellular enzymes are due to cell damage
65
enzyme activity can be measured through
- Measure initial rate
- Have substrate in excess
- Check that activity is proportional to enzyme concentration
66
In clinical samples, normal activity is
often given an arbitrary value
67
hexokinase is found in
muscle
68
glucokinase is found in
liver; aka hexokinase D
69
Isoenzymes can be studied by
electrophoresis as they are products of different genes.
| - sometimes alternative combinations ofdifferent gene products
70
Electrophoresis - a useful way to
separate plasma proteins
71
electrophoresis cathode
negative side - sample is placed here
72
electrophoresis anode
positive side - sample moves in this direction
73
Creatine kinase (CK) is a
dimer, made up from two polypeptides B and M
74
CK2 isoform is
abundant in the heart; elevation of plasma CK2 is diagnostic for myocardial infarction
Note: troponins I and T are now considered more useful
75
hexokinase catalyses the production of
glucose-6-phosphate from glucose and ATP
76
Catalysing a reaction with two or more substrates usually
involves transfer of groups from one substrate to the other
This can occur in several ways:
- Random order or Ordered with a ternary complex
- No ternary complex formation
77
Lactate dehydrogenase exhibits
```
an *ordered sequential mechanism*to its catalysis of pyruvate to lactate.
The coenzyme (NADH) binds first and the lactate is always released first,
```
78
In a ordered sequential reaction mechanism the enzyme exists in
a ternary complex, first with the substrates of the reaction and then (after catalysis) with the products of the reaction
79
Aspartate aminotransferase shows a
*double displacement* or *ping-pong* reaction pathway when it transfers an amino group from aspartate to α-ketoglutarate.
The term “ping-pong” comes from the fact the substrates bounce on and off the enzyme as they are catalysed. Aspartate “bounces” to oxaloacetate and α-ketoglutarate “bounces” to glutamate
80
Allosteric enzymes do not follow
M-M kinetics
81
Allosteric enzymes are made up of
many subunits, which contain many active sites.
One substrate binding to an enzyme subunit can cause changes in other active sites on other subunits
- This can lead to the concept of “cooperative binding” of substrate molecules
- Haemoglobin is a good example of cooperative binding of a substrate
82
What will affect an enzyme?
- Temperature
- pH
- Inhibitors
83
pH effects on enzyme
- pH changes the charge of amino acids
- If the active site amino acids charge changes the enzyme will cease to function correctly
- Extreme pH will denature most enzymes
- pH will also affect the substrates of the reaction, some of which may require H+ or OH- groups to be involved in the reaction
84
Competitive inhibitors bind to
enzymes non-covalently and will usually resemble the substrate molecule, therefore competing with the active site
85
competitive inhibition leads to
decrease in the affinity between the active site and the substrate, so the Km of the substrate-enzyme complex increases
86
how can you overcome competitive inhibition
Increasing substrate concentration can overcome this inhibition, so the same Vmax can be achieved
Therefore competitive inhibitors exhibit increased Km values but the Vmax remains unchanged – this gives a Lineweaver-Burke plot that look like line above original
87
(Azidothymidine) AZT acts by
competitive inhibition of the reverse transcriptase enzyme.
| - Reverse transcriptase is used by HIV to produce a dsDNA molecule from it’s ssRNA
88
AZT undergoes
triphosphorylation in the body, and thus mimics the ordinary DNA precursor thymidine triphosphate (TTP)
89
maximal interaction occurs between
enzyme and the transition state
90
In the body oseltamivir is
hydrolysed in the liver to it’s active form, which is then able to block the activity of neuraminidase enzyme.
- Neuraminidase normally cleaves sialic acid (found on the surface of cells) that allows the release of new virus particles from the cells
91
transition states are
intermediate step between an enzyme-substrate complex and an enzyme-product complex - difficult to replicate
92
naturally occurring catalytic antibody lupus erythematosus is characterised by
autoantibodies attacking the connective tissue of the joints, skin, kidneys, heart and lungs
93
Non-competitive inhibitors bind to
enzymes non-covalently and will usually attach to a site other than the active site of the enzyme.
The substrate is usually still able to bind the active site, so the Km of the substrate-enzyme complex remains unchanged
94
Non-competitive inhibitors and increasing substrate concentration
Increasing substrate concentration does not change the inhibition so the Vmax will decrease
- Therefore non-competitive inhibitors exhibit unchanged Km values but the Vmax decreases – this gives a Lineweaver-Burke plot that look like line above original
95
Irreversible inhibitors bind to
enzyme in a covalent, and therefore irreversible way
96
CN- binds to Fe3+ of
cytochrome c oxidase and disrupts the terminal respiratory system
- Blocking the terminal respiratory system will effectively “starve” cells of ATP causing the individual with cyanide poisoning to exit the carbon cycle post haste
97
often the first enzyme in pathway holds
regulatory step for that pathway – makes sense as you don’t want to regulate something half way down a pathway
98
Two main ways regulatory enzymes modulate reactions:
- Allosteric enzymes
- Covalently modified enzymes
Both classes of regulatory enzymes tend to be multi-subunit proteins
99
Allosteric effectors are usually
cell metabolites that bind non-covalently to a site on the enzyme that is not the active site
- This changes the enzymes structure
- Some effectors are activators and some are inhibitors
100
Allosteric effectors are examples of
non-competitive inhibitors
101
Allosteric enzymes :
| low [S] (between A and B)
sensitises the enzyme so it responds more efficiently at higher [S] (between B and C)
102
2 models explain allosteric enzyme kinetics
- Concerted model
| - Sequential model
103
Concerted model:
- Each sub-unit can exist in two different conformations
- One binds substrate well the other doesn’t
- With no substrate the enzyme flips between the two conformations
- All sub-units must be in the same conformation (so they flip in concert)
104
Concerted model explains the
sigmoidal curve
105
According to the concerted model, Allosteric activators will
- stabilise the ‘open’ conformation allowing S to bind more effectively
106
According to the concerted model, Allosteric inhibitors will
- stabilise the ‘closed’ conformation and make it difficult for S to bind effectively
107
Sequential model assumes
- No flipping between different conformation states
- Sub-units exist in a conformation that can bind S, activators, inhibitors
- It is the binding that causes a conformational change
108
In Sequential model, Substrate binding causes
a change in one sub-unit
- This causes a change in another sub-unit allowing it to bind S more readily
- Like the first model, binding of some substrate sensitises the enzyme to bind more
109
In Concerted model, Substrate binding causes
'locking' of the other binding sites, stopping them flipping, allowing other S to bind easily
- Low [S] sensitises the enzyme to bind more S
- Explains the sigmoidal curve
110
Many ways enzymes (and other proteins) can be regulated through
reversible covalent modification
| - most important covalent modifications is phosphorylation
111
Approx. 30% of all eukaryotic proteins are phosphorylated
- At a single site
- Multiple sites
- Multiple phosphorylations at one site
112
Enzymes catalyse the phosphorylation of enzymes
Protein kinases – add phosphoryl groups to proteins
| Protein phosphatases – remove phosphoryl groups
113
Multiple phosphorylation sites allow
very fine control of enzyme function depending on the requirement of the particular enzyme at a given time
- has finely tuned activity dependant on the signals it receives
114
Enzymes can exist as an inactive precursor protein, called
a proprotein or proenzyme
| - Proproteins can be cleaved to give active enzyme by proteases
115
zymogens - Proteolytic Cleavage
Digestive enzymes are regulated in this way – if they weren’t they would digest the parts of the gut where they are made
116
can insulin be cleaved
yes
117
Inhibitors can affect enzymes in different ways:
- Competitive inhibitor
- Non-competitive inhibitor
- Irreversible inhibitor
- Feedback inhibition
118
Feedback inhibition
caused by build up of something
119
Enzymes can be modulated in different ways:
- Allosterically
- Covalent modifications
- Proteolytic cleavage
120
Electrophoresis can be used as a
diagntstic tool to separate complex mixtures of enzymes
