Enzymes Flashcards
What are enzymes
biological catalysts
They increase the speed of specific chemical reactions
Not chemically changed by reation catalysed
Why are enzymes important
Because reactions like DNA synthesis, without enzymes, would take billions of years to occur
True or False?
The Majority of proteins are enzymes
True
But RNA enzymes also exist too
How can enzymes differ
In complexity e.g. how many subunits it has
How fast they operate
Where they are found
What reactions they catalyse
Enzymes have nomenclature
What do: oxidoreductase, transferase, hydrolase and lyase do?
Oxidise/reduce
Transfer chemical group
hydrolytic cleavage
Removal of a chemical group
Enzymes have nomenclature
What do: Isomerase, ligase and polymerase do?
Chemical rearrangement
Joining of two chemical
Polymer synthesis e.g. DNA
What is a substrate
The chemical that enzymes act upon
Substrate specificity is common, but doesn’t always occur
What is an Active site
A region of the ezymes that interact with the substrate
This close interaction allow the reaction to be sped up
Enzymes can bend, and have the ability to take on different configurations
Why is this an important characteristic of enzmes and how does it occur?
The tight, induced fit helps speed up the reaction
Charge on the active site, start to interact with charges on the substrate, and begin to pull electrons and bonds, which put the enzyme in an increased tension state. The likelihood of undergoing a reaction increases
Describe the enzyme cycle
- The substrate interacts with the active site of the enzyme, through diffusion and collision
- Once bound, the enzyme holds the substrate tighly, as the binding triggers a change in the enzyme conformation
- The bond between the two subunits begins to be put under tension and streched, causing the charges of the enzymes active site to act upon the charges in the substrate
- The enzyme is more likely to interact with water and split apart into its constituent pieces
- When it does so, the produces are released because the chemical configuration and balance of charges changes, which changes the configuration of the active site and the enzymes opens up
- The cycle can now begin again
Glucose is an important source of energy which the body breaks down using enzymes, without enzymes why would this reaction not at normal body temperature
Gluose has higher energy state, where it has more energy stored within it than in the products of carbon dioxide and water
The enzyme helps reduce this activation to go from gluose to its substituents
True or False?
Enzymes allow reaction to happen that would otherwise not occur
False
Most enzymic reaction can be done without enzymes
Reactions would happen without them, but slower
What is ‘inverting’ sugar and what are the two ways to do it
Hydrolysing polysaccharides and disaccharides into monosaccharides
Can be done through boiling or enzymatically
Industrially why would you use enzymes rather than boiling the invert sugar
Boiling: Easy, but energy expensive and industrially needs acid for effectivness
Enzymatic: Lower temperatures and doesn’t risk burning or colouring the sugar
Relating the the environment of where the enzymes are found, how can enzymes differ
Enzymes have optimal temperatures and pH ranges
This is because deviation from the optimum can affect the stability and speed of the enzyme
What is the issue with using enzymes as target drugs
They can be inhibited by other substrates
When using enzymes for certain drugs, what are the two ways they can be obtained
Human vs microbial protein in medical treatment
Sometimes recombinant enzymes can also be used
What is the benefit of using some enzymes in medical setting
They are extremely specific and can inhibit, or activate a certain reaction to occur
What are the benefits are using enzymes industrially
Allow reactions to occur at lower temperatures
Allow complex reactions to occur
Why may enzymes be used in the lab
To increase the sensetivity of assays
Copy and join pieces of DNA in PCR
Streptokinase is an enzymes which comes from a bacteria which acts as a clot-buster-dry
What is the issue with using it
As a foreign protein, it can induce immune reponse or allergy
Because of this, it has now largely been replaced by recombinant tPA (tissue plasminogen activator) which is a humanised enzyme product and is not recognised for foregin
Rennet is extracted from calves stomachs and is used to convert liquid milk to curds
What is the issue with this and how has it been overcome
Genetic modification of fungi allows production of vegetarian rennet - purified chymosin B
How have enzymes been used in cooking
Enzymes have been used to tenderise meats
What are biological washing agents
Enzymes which are added to help remove stains
Proteases, cellulases, amylases and lipases may be used
many are bacterial in origin
These enzymes are contains within a wax shell to protect them from the harsh environments before being put in the washing machine
Many drugs are enzyme inhibitors
There is two main classes: Reversible or irreversible
What is the difference between the two
Reversible - may be compete with substrate for active site, may also bind elsewhere and reduce function
Irreverisble - most covalently link to enzymes, often in/at active sites
Penicillin is an irreversible inhibitor
How does it work
Binds to bacterial transpeptidase active site and forms an ester linkage to the enzyme
Stops cross-linking of cell wall peptidoglycan
New cell wall is weak and fails, cell bursts
How have bacteria become resistant to penicillin
How can this be overcome
High-level resistance often is mediated by B-lactamases
These cleave the ring structure of penicillins
Clavuanic acids can enter the B-lactamase active site and covalently bond to it, it permently bond to the B-lactamase active site causing it to become inactivated
What is the issue with over or under expression of an enzyme
Can lead to cell dysfunction and disease
Hence enzymes are really important for the treatment and monitoring of diease
What is the substrate-binding site
Substrates bind to specific sites via interactions with the enzyme’s amino acids
Spatial geometry dictates specificity
What is the Active catalytic site
The region where the reaction occurs
Functional groups present include co-enzymes, metal ions, amino acid residues
What is the transition state
The high-energy intermediate which energy is need to form
Enzymes reduce the energy of this transition state
What are two ways to increase an enzymatically controlled reaction
Moving towards both optimum temperature and pH
How do enzymes affect equilibrium
Enzymes do not alter the position of equilibrium but rather accelerate its establishmentn
There is two models of substrate-binding
What are they and how do they differ?
Lock & Key: Complementary 3-D surfaces recognises the substrate, binds by forming bonds. Can be prevented by steric hinderance and charge repulsion
Induced fit: As substrate binds, the enzyme undergoes conformational change and side chains of amino acid active site reposition
What happens when the transition state complex forms in an enzymatic vs non-enzymatic system
Unstable high-energy complex with strained electronic configuration
‘activation energy’ of formation is reduced compared to the non-catalysed reaction
What happens once the transition state is formed
The transition state is the point at which bonds become maximally strained
It decomposes to products
Enzyme returns to its original form and can bind to new substrates
What is activation energy and how does it affect the overall rate of reaction
Activation energy is the difference between substrate and transition-state complex
The overall rate of reaction is determined by no. of molecules aquiring the activation energy
Enzymes decrease the activation energy
What are co-factos/co-enzymes
Needed for enzymes to work
Catalytic properties often dependent on non-peptide molecules
NAD+, NADP+, FAD
TIghly bound cofactors are know as prosthetic groups: Mg2+, Fe2+, Zn2+ (often to stabilise negative charges)
If a singular enzyme becomes non-function, why is this problematic on a larger scale
Some enzymes promote consecutive reactions in a metabolic pathway
Associate to form a multi-enzyme complex
What are the benefits of multi-enzyme complexes
Transit time via diffusion is reduced
Less interference: products are acted upon by correct enzyme
What are Isozymes
Enzymes can catalyse the same reactions as one another, even if the majority of their sequence differs, due to interactions being due to 3D spatial arrangement
They can catalyse the same reactions but will have different properties to them e.g. different speeds of conc of substrate
In terms of enzyme kinetics, what does the value of k stand for
What is the ‘stead state’ in an enzyme catalysed reaction
After the product formation has gradually built up and the reaction rate and intermediate concentration changes relatively slow
How can you compare enzyme
By comparing how fast they are during the inital rate of reaction (V0)
What can affect the value for V0
The amount of substrate or enzyme present
pH, temperature, activators and inhibitors
For most enzymes, inital rate varies hyperbolically with substrate concentration for a fixed enzyme concentration
This is expressed through which graph and equation
Michaelis-Mention Equation
What is the Michaelis constant
Is the concentration of substrate that the enzyme is working at half maximal velocity (Vmas / 2)
This is how scientists will often compare enzymatic behaviour
On the Michaelis-Menten curve, what happens as we approach Vmas on the curve
We seen zero order or saturation kinetics
You reach a level of substrate, where if you add anymore the enzymes will not increase anymore in speed
On the Michaelis-Menten curve, was is happening at the start of the curve
We see something called first order kinetics
You have lots of enzymes present with active sites which are not occupied
So as substrate levels are increased, the rate also increases in a proportional fashion
What is the benefit of knowing Km of an enzyme
In many cellular environments, the concentration of substrates present in that environment are in the vicinity of Km
Hence by tweaking the amount of substrate present, e.g. a decrease would lead to a significant decrease in speed of enzyme, however an increase would only lead to a minor increase is speed
What is Vmax
The maximum operating speed of your enzyme
(This can change depending on how much enzyme is present)
True or False?
Km can change like Vmas
False - Km is the Michaelis constant
Km is the substrate concentration at which the initial rate is 1/2 Vmax
What is the benefit of knowing Km
Permits calculation of the substrate required to saturate all active sites
What is the effect of an enzyme having a low Km value
If you hav an enzyme with a low Km, it means it only needs a small amount of substrate present before it reaches half maximal velocity - high substrate affinity
What is the effect of a enzyme having a high Km
In order to reach 1/2 Vmax, you need a large amount of substrate present
This would indicate the enzyme has a low affinity for the substrate
Kd is the dissociation constant
What does it measure
How likely the enzyme is to break apart
What does Kcat measure
The rate constant of the conversion of the enzyme substrate complex to product
Basicaly measures catalytic efficiency (n)
High catalytic turnovers are only realised at high substrate levels and rarely achieved in cells
What is the limit on catalytic efficiency
How have some enzymes overcome this
For a substrate to be converted to products, the substrate and enzyme molecules must collide and randomly diffuse together
This has a theoretical limit of about 10⁹ M¹ s¹
Yet some enzymes such as acetylcholine esterase and catalase approach this value through: multi-enzyme complexes and embedding in cell membranes
Before the updating of modern computers, it was difficult to work out what Vmax was (hence what Km was) because the Michaelis-Menten curve is an asymptote
How was this overcome?
The equation for the curve, could be rearranged and linearised
The curve can be converted into a straight line graph (forming the Lineweaver-Burk plot)
How can you use the Lineweaver-Burk plot to work out Km and Vmax
What are the drawbacks of using the Lineweaver-Burk plot to work out Km and Vmax
Becaus of the way it is calculated it have a very unequal distribution of points
It hav emphasis the points with low substrate concentration (prone to experimental error)
What is 1 unit of enzyme activity (U)
The amount of enzyme which transforms substrate into product at an initial velocity of one micromol per minute measured under defined assay conditions
What is the specific activity of an enzyme
Enzyme activity per mg of protein
What is an inhibitor
Something that can affect enzyme activity by binding to the active site
If a compound isn’t part of the normal reaction they are an inhibitor
Works by decreasing the reaction velocity
Why is it useful to understand and classify inhibitors?
- Insight into catalytic mechanisms
- Insight into metabolic control
- Permits synthesis of inhibitors as therapeutic agents
What is a reversible inhibitor
Is not covalently bound to the enzyme
Can be removed by dialysis
Characterised by Ki (inhibition constant) - how much inhibitor to block 50% of the active sites
What is a competitive inhibitor
A substance with a very similar structur to the substrate
Hence competes with substrate at substrate recognition site
Can be overcomeby increaing the substrate concentration
What affect will a competitor have on Km and Vmax
Inhibitors increase the apparent Km (lower affinity of the enzyme)
No affect on Vmax, as if you keep increasing the amount of substrate Vmax for with and without the inhibitor will be the same
How would the lines on the Michaelis-Menten and Lineweaver plot change if a reversible inhibitor is added
What is a non-competitive inhibitor
Does not compete with a substrate for its binding site
What effect does a Non-competitive inhnibitor have on Vmax and Km
As the substrate cannot bind due to the non-competitive inhibitor binding in a seperate site, Vmax is lowered
The amount of active (unaffected enzyme) in the system is lowered)
Km stays the same as the unaffected enzymes will still reach half Vmax at a certain concentration of substrate
How is the Michaelis-Menten and Lineweaver-Burk plot affected by non-competitive inhibitors
What is an Uncompetitive inhibitor
Can only bind the the enzyme-substrate complex (ES) and not to free enzyme
Inhibitor binding must be located either at a side created by conformational change or to substrate
Creates a dead-end complex (ESI)
Cannot be overcome by increasing the substrate concentration
What impact does uncompetitive inhibitors have on Km and Vmax
Vmax is decreased because the substrate cannot unbind
Km decreases because when these inhibitors bind the the E/S complex they increase the affinity of the enzyme to the substrate
How do uncompetitive inhibitors affect the Michaelis-Menten curve and the Lineweaver-Burk graphs
How can substrate act as inhibitors
The substrate of some enzymes can act as an uncompetitve inhibitor
At very high substrate concentrations, substrate binds to second non-catalytic site
How may the end-product of an enzymatic pathway affect enzyme activity
Why is this important
‘product inhibition’
Important in metabolic pathways
Prevents one enzyme from swamping the next enzyme in line
First enzyme in unbranched pathways are effected by the final products
In branched pathways, the first enzyme after he branch point are affected
What are irreversible inhibitors
Bind to enzyme via covalent bonds (though not all bind covalently, tight non-covalent is enough)
Cannot be removed by dialysis
Reduces the amount of enzyme available for reaction
Can target functional groups or metal ion active sites
What are suicide inhibitors
There are unreactive until they bind to the active site of the enzyme
Undergo several steps of the reaction and the converted into a more reactive compound, then combine irreversibly
Why is it important to regulate enzymes within the body
In the human body thousands of enzymes work, at any one time, to ensure efficient metabolism and physiological function without waste (e.g. time + energy)
Enzymes must be regulated - increased or decreased actvitiy - to maintain metabolic effectivness
More enzymatic reaction operate close to equilibrium, knowing this, how can we regulate these reactions
The slowest (‘rate limiting’) step of a metabolic pathway is the most efficient control point of pathway
So you can regulate enzymes at that point
Substrate reponse and product inhibition are 2 basic methods of control
Why is substrate reponse a good form of regulation
When can this not be a good form of regulation
Many substrates exist at the value of Km
Hence if the amount of substrate was to decrease by a small amount, the velocity of th enzyme would significantly decrease
May not always be effective with long, divergent pathways
Rate-limiting enzymes are controlled by …
By mechanisms that affect the catalytic site
What are the 4 main reglatory mechanisms of enzymes
- Allosteric activation/inhibition
- Phosphorylation (or other covalent modification)
- Protein-Protein intractions
- Proteolytic cleavage
What is Allosteric Regulation
Activity Modulation via reversible, non-covalent binding of small molecules called ‘effectors’
They bind at allosteric sites, not active sites or substrate binding-sites
Allosteric effector binding changes catalytic site conformation which affects substrate binding
Process is rapid, and the first response of cells to condition changes
What feature do enzymes usually have if they are affected by allosteric regulation
Often composed of multiple subunits
And the allosteric site is often not on the catalytic subunit
What are the advantages of Allosteric Regulation
- Effectors, as they bind to sits other than a catalytic site, can be activators (not just inhibitors)
- Effectors do not need to resemble substrate or product
- Regulation is rapid
Allosteric regulators can either be allosteric activators or inhibitors
What does this mean?
Allosteric activator = Effector binds and enzyme activity increases
Allosteric inhibitor = Effector binds an enzyme activity decreases
It is important to note that in both these cases Vmax is not changing but Km is (affinity of the enzyme)
What is K0.5
Used in terms of allosteric enzymes
The concentration of which you have half of the active sites on the enzyme saturated
What is a Hetrotrophic effector
Compounds that are different from the substrate which serves as an allosteric effector
Sometimes you will find the substrate itself can bind to an allosteric binding site on the enzyme and change its activity
These are known as
Homotrophic effectors
Not all but large majority of allosteric enzymes have a property called cooperativity, what is it?
Usually, a homotrophic effector functions as a positive effector …
When a substrate binds to one subunit it can enhance the catalytic properties of the other subunits - ‘positive co-operativity’
However, the opposite can also occur and a reduction in catalytic properties of other subunits can also occur - ‘negative co-operativity’
What is the ‘Concerted Model’
There is a T-state and a R-state
The T-state is the tensed state and the R-state is the relaxed state
These two states have different binding affinities for the substrate
In the model, your enzymatic complex exists the majority of time in the T-state
However, when your effector binds to the allosteric site, the enzymes shift from T-state to the R-state
Now all the enzymes have a enhanced ability for binding to the substrate
What is the sequential model
When a allotrophic effector binds, you get not only one of the subunits changing into th R state, but another subunit will also change into the R state, then another subunit etc
The R-state will have an enhanced ability for binding the substrate
Name some real life examples of allosteric regulators
Many key enzymes in metabolism that fuel oxidation pathways are allosteric enzymes
e.g. Glycolysis (phosphofructokinase)
e.g. TCA cycle (isocitrate dehydrogenase)
A type of covalent modification is phosphorylation
What occurs during this process
- The addiaition of a phosphate groups on serine/threonine/tyrosine residues
- Due to the bulky and negeative native of the phosphate it can alter ionic interactions and hydrogen bonds nerby
- It can impact protein structure and hence function of the enzyme
- It is important to note that just because an enzyme becomes phosphorylated it becomes active as it depends on the pathways you are dealing with
How is protein kinase and protein phosphatases used in phosphorylation?
Protein kinase: sticks phosphate groups on the proteins
Protein phosphatase: removes phosphate groups via hydrolysis
Protein kinase can differ in function, how?
Some protein kinases only regulate one protein, others simultaneously regulated several rate-limiting enzymes
Protein kinase A - is a serine/threonine protein kinase which phosphorylates several enzymes that regulate different metabolic pathways
Adrenaline increases intracellular concentration of 3’-5’ - cyclic AMP, how?
What does the cAMP then do?
Protein kinase A responds to the activity of adrenaline
This will be detected by a receptor in the membrane and lead to the creation of cAMP
cAMP will bind to the inactive protein kinase A and cause regulatory subunits to fall off and protein kinase to full work
Phosphorylated forms may be more or less active than unphosphorylated forms
In the terms of glycogen synthase explain this?
Also, other modifications can affect the ability of the enzyme to interact with other proteins or to reach its correct locations?
Phosphorylation of glycogen synthase leads to a decrease in its activity
Glycogen phosphorylase (removal of a phosphate) increases its activity
The addition/removal of acetyl, ADP-ribose, or lipids
How can protein-protein interaction affect enzymes
Direct interactions by proteins can lead to a conformational change in the active site
Name an important example of protein-protein interactions as a form of regulation
The calcium-calmodulin family of modulator proteins
These will reponse to the presence of calcium, which will cause a change in its own structure and can now bind to its targets
How does the calcium-calmodulin modulates the activity of glycogen phosphorylase kinase (enzyme that sticks a phosphate on glygen phosphorlase)
Glycogen phosphorylase kinase has a calmodulin subunit, which will allow it to respond to calcium release in the muscle (if a nerve impulse hits the muscle and calcium is released from the sarcoplasmic reticulum)
This will lead to a conformational change and the calmodulin will hug the enzyme slightly differently and the ezyme will becolme activated
Another example of protein-protein interactions for regulation is the Monomeric G proteins
These are GTPases and will bind to Guanosine Triphosphate and hydrolyse it forming GDP
Once they have bound, they become inactivated as a conformational change occurs
GAP proteins can also encorage GTP to be broken down quicker and GTPases will inactivate quicker
How do guanine exchange factors affect GTPases
They will swap GDP for GTP basically activating the proteins
And Guanine dissociation inhibitors which will prevent GDP from leaving the G protein and lock them in an inactive state
How can proteolytic cleavage control enzymatic activity
This causes the enzyme to basically be chopped up
Enzymes can be irreversibly activated or inactivated (zimmerogens) by proteolytic enzymes
Degradation of enzymes by intracellular proteases by lysosomes or proteasomes determines enzymatic activity over a much longer period
