Session 4.4a - Lecture 2 - Enzymes Flashcards
Slides 1 - 28
Enzyme activity: kinetics and inhibition
- Role of enzymes
- How enzymes work
- Why they’re important
- How we can study enzymes
Enzyme activity:
- kinetics
- measurement
- inhibition
- measuring inhibition
Role of enzymes (start to introduce what proteins do)
Answer these for ILO.
What are enzymes?
Biological catalysts that increase the rate of chemical reactions by lowering the activation energy.
What is a chemical reaction simplistically?
A chemical reaction is where we’re starting with a substrate and ending with a product. In many cases this can be REVERSIBLE, we can go in the opposite way.
Which normally has lower energy - the substrate or the product?
The product
What is the energy change for reactions commonly measured in?
Gibbs free energy (the change in free energy)
What causes the energy change in substrate to product creation?
Making and breaking bonds
When we look at energy diagrams there is a barrier we need to get across. What is this known as?
The activation energy
What is the activation energy?
The minimum amount of energy a substrate molecule must have to form our product.
“Minimum energy S must have to allow reaction”
What is the transition state?
The state of a chemical midway between the substrate and product.
“High energy intermediate that lies between S and P”
(e.g. think about breaking a stick in two - the original stick is the substrate; when it is flexed in half to break it is the transition state; the final two pieces are the product)
Draw a simple chemical reaction.
S P
Fig. 2
Label this graph.
x-axis: Progress of the reaction –>
y-axis: Free energy, G
beginning: S Ground state
end: P Ground state
middle: Transition state (+)
- -> delta G+ S–>P (how much energy it takes to go from S to P)
- -> delta G+ P–>S how much energy it takes to go from P to S)
Draw an energy diagram for an enzyme.
See Fig. 2
x-axis: Progress of the reaction –>
y-axis: Free energy, G
beginning: S Ground state
end: P Ground state
middle: Transition state (+)
- -> delta G+ S–>P (how much energy it takes to go from S to P)
- -> delta G+ P–>S how much energy it takes to go from P to S)
What happens to the rate of chemical reaction without a catalyst?
The rate of chemical reaction will not be very fast as not many molecules will have the activation energy.
How can you increase the rate of reaction?
- Temperature
2. Concentration
Why does increasing the temperature increase the rate of reaction?
It increases the number of molecules with activation energy
- We give the molecules more energy so more of them are more likely to have the activation energy so they can overcome this intermediate state.
Why does increasing the concentration increase the rate of reaction?
Increases chance of molecular collisions
- pack in more molecules, if there’s more of them there’s more chance of them interacting so more chance for reaction to occur
Why is it difficult to use temperature to increase the rate of reaction in humans?
If I took you and heated you up to 50 degrees, your reactions might occur faster, but then you’d also be dead …
Why is it difficult to use concentration to increase the rate of reaction in humans?
E.g. glycolysis
- usually a few mM glucose in the cell
- could whack it up to 1 M glucose
- be much faster BUT
- much more serious problems because cells would explode
Why do cells explode if you take in too much glucose?
Because the cells would take in so much water and hence explode
Why can’t we change temperature or concentration to increase the rate of reaction in a biological system?
The cells/you would die due to HOMEOSTASIS
What is the idea of homeostasis?
To keep things as constant as possible (yes, there are small changes in concentration, metabolism etc., but not by many orders of magnitude).
If we can’t increase the rate of reaction by temperature and concentration, how can we?
By binding catalysts, or enzymes.
How do enzymes increase the rate of reaction?
They lower the activation energy (catalysed reactions over uncatalysed reactions)
What does lowering the activation energy do in reactions?
It means more molecules are likely to have enough energy to react - this is the basis of what an enzyme it.
How does an enzyme lower the activation energy?
- By facilitating the formation of a transition state
- By bringing together molecules, e.g. if you get two reactants and you want them to react then by bringing them physically closer together, we increase
Fig. 3
Label this image
x-axis: Reaction progress –>
y-axis: Free energy –>
Substrate (beginning)
Transition state, S+ (hump/peak)
Product (end)
delta G+ (uncatalysed) (from S to higher S+)
delta G+ (catalysed) (from S to lower S+)
delta G for the reaction (substrate to product)
Draw the energy diagrams for a catalysed and uncatalysed reaction.
See Fig. 3
x-axis: Reaction progress –>
y-axis: Free energy –>
Substrate (beginning)
Transition state, S+ (hump/peak)
Product (end)
delta G+ (uncatalysed) (from S to higher S+)
delta G+ (catalysed) (from S to lower S+)
delta G for the reaction (substrate to product)
Reaction is the same but catalysed transition state must be LOWER than the uncatalysed transition state.
What are 6 important features of enzymes?
- Highly specific
- Unchanged after the reaction
- Do not affect the reaction equilibrium
- Increase the rate of reaction
- Proteins
- May require associated cofactors
Explain the phrase “highly specific” in relation to enzymes.
Most enzymes will only react with one certain substrate - they are very fussy and will not go after lots of different things.
What is the clinical significance of an enzyme being highly specific?
Normally, enzymes will only react with one substrate.
In some cases they will react with other substrates if things go wrong in the body, and you’ll do that in some units you do later, e.g. in galactosaemia for metabolism
Explain the phrase “unchanged after the reaction” in relation to enzymes.
Biological catalysts are unchanged after the reaction – might change during the reaction, might bind things, might move things around, often move protons in there - but at the end, enzyme will be same as the start
Identify four ways an enzyme can change DURING the reaction.
Enzyme might:
- change during the reaction
- bind things
- move things around
- often move protons in there
but ultimately, will remain unchanged at the end.
Explain the phrase “do not affect the reaction equilibrium” in relation to enzymes.
If the enzyme is unfavourable for certain enzymes it WON’T make it more likely to occur, it only increases the RATE.
Enzymes increase the rate of reaction.
What is a critical thing to remember about this?
They increase BOTH the forward and reverse reaction.
Are all enzymes proteins?
Most enzymes are proteins; not every but almost all, thus, virtually all enzymes are proteins.
What are cofactors?
Some enzymes may require associated help, from cofactors and coenzymes, to help them work, e.g. in metabolism.
Why are we interested in enzymes?
- Inheritable genetic disorders
- Overactive enzymes can cause disease
- Measurement of enzyme activity for diagnosis
- Inhibition of enzymes by drugs
What is the clinical significance of enzymes in inheritable genetic disorders?
Many mutations we see in inheritable genetic disorder often affect enzymes, e.g., many inheritable metabolic diseases have mutations involving enzymes.
What is the clinical significance of enzymes and disease?
By changing enzyme function (via mutation) you can cause disease.
How can overactive enzymes cause disease?
By switching on enzymes that are not normally present or at a certain level you can change the activity of an enzyme, which causes disease.
having an understanding of these enzymes and measuring them important for understanding disease properties.
Give a clinical example of overactive enzymes causing disease.
Cancer - signalling molecules have enzymes involved - things like tyrosine kinases are upregulated in many aspects of cancer.
What can the measurement of enzyme activity be used for clinically?
- important for understanding disease properties
- for diagnosis, e.g. via reference ranges.
What can the measure of enzyme activity tell you about the enzyme itself?
- Whether it’s being affected
- Whether an enzyme is in the right place - this will tell you whether the tissue is being damaged or not.
How do drugs target enzymes?
Many diseases are treated by drugs that target enzymes by acting as inhibitors.
What is the active site?
The active site of an enzyme is the place where substrates bind and where the chemical reaction occurs
(critical part of enzyme that does anything, point where substrates bind and reactions occur)
Describe the active site in relation to the size of the enzyme.
Active sites only occupy a relatively small part of the protein molecule (enzyme) itself
How many amino acids are active sites?
Most enzymes are >100 aa but active site is only a few aa
E.g. lysozyme, active site is only made up of ~6 amino acid residues (AAR) out of 129 AAR proteins (that’s quite a lot - some proteins are enormous but active site might be only a few amino acids)
Active sites only occupy a relatively small part of the enzyme itself.
Why do we need the rest of it then?
Most of enzyme acts as a scaffold to create the active site - enzyme works together to form a unique 3D shape to bring the active site together
Which amino acids in a sequence form the active site?
The active site is formed by amino acids from different parts of the primary sequence - so even though these residues are spread throughout the molecule, physically they’re close - forming an active site region where we can get binding of the substrate..
Fig. 5
Label and caption this diagram
Lysozyme
N 1 35 52 62,63 101 108 129 C
129 amino acid residues, 6 amino acid residues (numbered) form the active site.
(would not need to draw)
What shape are active sites?
Active sites are often clefts or crevices
Where is the active site of an enzyme?
Usually not on the surface of the protein, but more buried away
What is the significance of active sites being clefts or crevices?
Substrate molecules are bound in a cleft or crevice that usually
excludes water
Substrate molecules have to go into a cleft or crevice - important because defines what can get in, but in many cases used to exclude water
Substrate molecules are bound in a cleft or crevice that usually excludes water.
Why?
Water is so abundant and present at such high concentrations that it can interfere with many chemical reactions. It is important to exclude water in many cases, hence why you get active sites in these unique shapes (cleft/crevices).
What is the relationship between the shape of the active site and the substrate?
Active sites have a complementary shape to the substrate
What enzyme theory came out at the first half of the 20th century explaining active site complementarity?
“Lock and Key” hypothesis
What is the “Lock and Key” hypothesis and what is it’s potential problem?
Idea that the active site is complementary in shape to the substrate – substrate can come in and bind – then get a enzyme-substrate complex which then can be released.
But if a substrate is coming in and binding to the enzyme, why would it want to let go again - so it doesn’t sort of fully make sense.
Fig. 6
Label the image
Substrate + Enzyme Active site a b c –> ES complex a b c
Draw a reaction between an enzyme and a substrate from the “Lock and Key” hypothesis.
See Fig. 6
Substrate + Enzyme Active site a b c –> ES complex a b c
Substrate shape must be complementary to the enzyme, and fit into the enzyme perfectly to form an ES complex.
What is the “Lock and Key” hypothesis?
The active site of the enzyme is complementary in shape to that of the substrate.
After the “Lock and Key” hypothesis, what was the hypothesis used to explain enzyme-substrate binding?
“Induced fit” hypothesis
Does the “induced fit” hypothesis follow the rule that active sites have a complementary shape to the substrate?
The active site is complementary, but it only occurs after the substrate binds.
How can the “induced fit” hypothesis work alongside the “lock and key” hypothesis?
In the “induced fit” hypothesis, the active site is complementary, but it only occurs after the substrate binds.
This means there is some sort of complementary – e.g. particular amino acid residue that will bind substrate and only become important once physically bound – giving you “lock and key” hypothesis
Explain what we currently believe to be true about the active site of enzymes and their substrates.
Active sites have a complementary shape to the substrate
“Lock and Key” hypothesis
Binding of substrates can induce changes in the conformation
“Induced fit” hypothesis
When is the active site complementary to the substrate following the “induced fit” hypothesis?
The active site only forms a complementary shape AFTER binding of the substrate
Fig. 7
Label the image
Substrate + Enzyme a b c –> ES complex a b c
Draw enzyme-substrate binding in the “induced fit” hypothesis.
Substrate + Enzyme a b c –> ES complex a b c
Enzyme has a non-complementary but similar shape to the substrate. It becomes complementary after binding of the substrate.
How are substrates bound to the active site?
Substrates are bound to enzymes by multiple weak bonds - non-covalent interactions.
Why do substrates bind to enzymes with NON-covalent interactions?
We generally don’t want them to form strong covalent bonds – covalent bonds then they won’t get away again, so binding must not be too tight!
Substrates tend to bind to active sites through multiple weak bonds.
How can this be exploited pharmacologically?
Substrates don’t form strong bonds with active sites (such as covalent bonds) - otherwise they wouldn’t be able to detach.
However, inhibitors can be made which attach covalently to active sites. This means they effectively block the active site because the strong covalent bonds mean that they won’t leave the active site; so the substrate can’t attach (thereby inhibiting the active site).
How does uracil interact with ribonuclease?
Uracil, from a substrate, can bind to the enzyme ribonuclease. The side chain uracil makes several interactions with the active site residues from the ribonuclease enzyme.
What stabilises a uracil side chain when it interacts with a ribonuclease active site?
Hydrogen bond interactions from serine and threonine that stabilise uracil binding within active site, holding it in there whilst reaction is occurring
Fig. 8
Label the image
Uracil (from substrate)
Serine side chain
Threonine side chain
e.g. ribonuclease
(would not need to draw)
How do enzymes help form the transition state?
Enzymes hold the substrate in place to form the enzyme-substrate complex - this puts the substrate in the right place, forming the enzyme-substrate transition state, allowing the product to be formed.
How do enzymes help the substrate form a product? Describe using a parent-baby anology.
Baby (substrate) is wearing a jumper but wants to take it off because it’s too hot. By itself, the baby is very uncoordinated and flails around. It could take the jumper off if its arms are in the right direction but it needs a bit of help.
Our parent (presumably) is the enzyme which is able to hold the baby (S) in the right conformation, forming our ES complex.
The parent can guide the baby’s arms into the correct position to get the jumper off, to form the ES transition state. This is the nice state where the jumper can be taken off.
The jumper is taken off the baby (baby minus jumper) to form our product. Happy baby.
Fig. 9
Label this image
Substrate (S) –> Enzyme Enzyme-substrate complex (ES) –> Enzyme Transition state –> Product (P)
Graphs: x-axis: Progress of reaction --> y-axis: Free energy curve - S (beginning) - ES (dip) - T* (peak) - P (end)
Draw an energy diagram explaining how enzymes work to lower activation energy.
See Fig. 9
x-axis: Progress of reaction --> y-axis: Free energy curve - S (beginning) - ES (dip) - T* (peak) - P (end)
- S is the substrate
- Enzymes holds substrate to form ES complex, which lowers activation energy
- This allows T* (transition state) to form, which is the peak activation energy
- Leaving with a product, P, at the end, with the lowest activation energy
What do we mean by enzyme kinetics?
This is really just measuring the activity of enzymes.
What happens to an enzyme as a chemical reaction occurs?
If we think simplistically about an enzyme and what’s going to happen as a chemical reaction occurs, need to think about in terms of concentration of substrate, product and amount of time that’s passed by.
Why is T=0 important in enzyme reactions?
If we prepare a reaction (we’ve put some substrate in a tube and put it in the right pH, right buffer) and add some enzyme at time 0, then this is the only point we actually know how much substrate there is - we know the substrate concentration because we put a defined concentration of substrate in.
Why is T=0 the only point we actually know how much substrate there is in a reaction?
We know the substrate concentration here because we put a defined concentration of substrate in. However, this changes as a reaction progresses
What happens to the concentration of substrates as the reaction progresses?
Enzyme will work and chemical reaction will occur and more of our substrate will turn into product, thus, over time, more product will appear.
How can enzyme reactions be measured?
By measuring the appearance of product over time
(substrate turns into product with time in a chemical reaction).
We can also measure substrate disappearing as well.
How do product-time reaction graphs tend to look?
Tend to see sort of curve showing reaction slowing over time – amount of substrate decreases over time and product increases.
What is V0?
The initial velocity or initial rate that is drawn as a tangent to the product-time curve.
Why is V0 important?
Only time we actually know concentration of substrate is right at the start – so V0 is drawn as a tangent to this curve to work out the rate of reaction: change in product over time.
Fig. 10
Label this image
T=0 - all substrate, no product (and enzyme)
T=10 - some substrate, some product (and enzyme)
T=20 - less substrate, more product (and enzyme)
Graph:
x-axis: Time –>
y-axis: Product –>
Tangent = V0
V0 = initial rate of reaction
Draw a diagram demonstrating enzyme kinetics
Fig. 10 (top)
T=0 - S S S S S S S S E --> T=10 - S S S S S P P P E --> T=20 - S S P P P P P P E
Draw a graph demonstrating enzyme kinetics
Fig. 10 (bottom)
x-axis: Time –>
y-axis: Product –>
Tangent = V0
V0 = initial rate of reaction
curve shape - hyperbola
How do enzyme kinetics vary with different properties?
Enzymes show different kinetics with different properties, e.g. temperature, pH.
What sort of curve does an enzyme make when looking at kinetics of different properties?
A bell-shaped curve, with a maximum in the middle and reaction rates falling off sharply on either side.
How does temperature affect enzymes in your body?
The maximal rate of reaction will be at 37oC, or body temperature.
The rate of reaction will dip if the temperature is lowered/increased either side of this - this in your body is not going to make too much difference because the enzymes are not really going to see much difference from around 37oC.
How does temperature affect non-human enzymes?
Other enzymes in different organisms have a different temperature maximal rate:
- an enzyme taken from hot springs bacteria (important in PCR for thermostable polymerases) can be optimally active around 95 oC.
- enzyme taken from a marine bacterium – sea temp normally around 4oC, cyrophilic (extremophilic organisms that are capable of growth and reproduction in cold temperatures) bacteria, temp optimum around about 4-5 oC. (Normally a range)
How is temperature used for enzymes involved in PCR?
An enzyme taken from hot springs bacteria (important in PCR for thermostable polymerases) can be optimally active around 95 oC.
What is normal sea temperature?
Sea temp normally around 4oC
What is a cryophilic organism?
Extremophilic organisms that are capable of growth and reproduction in cold temperatures
How does pH affect enzyme kinetics?
Like temperature, you’d see a bell-shaped curve.
What is the optimum pH for most enzymes in the human body?
Most human enzymes have a pH optimum about 7.4, bc this is the pH you’d see in most of your tissues or cells or organelles (not completely true bc certain organelles more acidic, so there might be enzymes that optimise for that)
