Enzymes Flashcards
Recall the functions of enzymes
- Digestion- carbohydrates, fats, proteins
- Blood clotting- fibrin clot catalysed by thrombin ·
- Defence-immune system activation of complement
- Movement: muscle actomyosin is an ATPase
- Nerve conduction: membrane ion pumps for Na+, Ca2+
Recall the types of enzymes and their general functions
- Proteases- have proteins as their substrate
- Nucleases- cleave to phosphodiester bonds 3.Polymerases- simple building blocks to polymerises make
- Kinases- transfer phosphate from ATP unto a substrate eg hexokinase transfers a phosphate onto glucose.
Catalyse a particular type of reaction
What are the properties of enzymes? (4)
- Increase reaction rate by up to 10 billion fold by reducing the free energy needed
- Show specificity
Unchanged at end of reaction - Do not alter reaction equilibrium
Explain the difference between free energy of activation of catalysed and uncatalyzed reactions
- The substrate has a higher free energy than the product, so it releases free energy as the reaction goes on which makes it favourable reaction.
· Free energy must be put in to get form the transition state which is called the energy of activation which enzymes reduce
What is the transition state?
The transition state is the point at which if you go backwards you go to the substrate and if you go forward, you go the products.
What is the active site?
The active site is a 3-D cavity or cleft that binds substrate(s) with specificity through electrostatic, hydrophobic, hydrogen bonding and van der Waals interactions.
Where does evidence of the active site come from?(3)
- X-ray crystallography
- As the electron is accelerated, it releases X-rays of similar wavelengths and they can be read off and used to image the structure of the protein
- Kinetic studies of enzyme activity
Explain the lock and key theory
The substrate has a shape that is directly complementary to the enzyme and fits like a lock in a key, this model explains the specificity of the reactions.
Explain the induced fit model and give an example of an enzyme that uses this model
- states that the enzyme is flexible as the substrate starts to bind, the active site changes shape to fit the substrate more closely.
- This distorts the substrate molecule in the active site, making it more likely to change into the product.
- An example of an enzyme that uses induced fit is the glycolytic enzyme hexokinase which binds to glucose
Give examples of how induced fit speed up reactions(2)
If a bond in the substrate is to be broken, that bond might be stretched by the enzyme, making it more likely to break.
1. if a bond is to be made between two molecules, the two molecules can be held in exactly the right position and orientation and “pushed” together, making the bond more likely to form.
Where does evidence from induced fit come from?
From crystallizing enzymes before and after being bound to the substrate to see if the shape has changed
Recall how active binding reduce activation energy (7)
- To bring molecules together in the active site A + B = C + D an enzyme can bring together two reactants which is called approximation and this can speed up the reaction.
- To constrain substrate movement
- To strain bonds in the substrate making breakage easier. Substrate is distorted on binding to resemble transition state. So less free energy to get to the transition state
- To stabilise positive and negative charges in the transition state
- To exclude water from the active site- make reaction go faster- by enveloping the substrate
- To provide a reaction pathway of lower energy e.g. involving covalent enzyme-substrate intermediates. Break reaction down into smaller step with each step having lower free activation energy
- Use cofactors: bring new chemistry to the active site with NAD(H), FAD(H2), metal ions such as Mg2+
What is the Vmax?
The Vmax is the maximum possible velocity when all active sites are occupied
What is Km?
- Km is the measure of ‘stickiness’ of the active site for the substrate, so if the substrate is very sticky you don’t need to put a lot in to have Vmax.
What does low Km mean?
A low Km means a higher affinity or a stickier substrate because you need less to achieve the same V max.
What does the curve of the Michealis graph show?(2)
- The curve shows saturation behaviour as it increases steeply but then begins to level off and plateau
- the active sites are the limiting factor as there is no increase in rate.
Why is the Lineweaver-Burk equation more accurate?
V max does not have to be estimated it can be calculated
How do you calculate the turn-over number?
The turnover number is V max(max no of substrate molecules handled) divided by the amount of enzyme(no. of active site) there is.This is the Kcat.
How do you plot a double reciprocal polt?
Measure the rate of product formation at various substrate concentration.Plot 1/V against 1/S. Intercepts on the axes provide values for Vmax and Km
What happens to Km and Vmax in the presence of a competitive inhibitor?
· Km is increased (it takes more substrate to achieve Vmax/2).However, Vmax is unaltered as the effects of the inhibitor can be competed out at high substrate concentrations.Increasing amounts of substrate can swamp the inhibitor (present in fixed concentration), allowing the enzyme to effectively not see the inhibitor at high substrate concentrations.
What happens to Vmax and Km in the presence of a non-competitive inhibitor?
· Vmax is reduced. There is always a fixed amount of enzyme inactive in non-competitive inhibition. change the amount of enzyme, you change the Vmax.
2. Km does not change. There are less active sites available but the affinity of the substrate to the active site has not changed.
Why is it important that enzymes are regulated?
If all enzymes are working at full capacity then everything is converted into products and that is not helpful as there will be no more substrates
Summarise the ways in which enzymes are regulated(4)
- Control of gene expression-enzyme amount
- Compartmentation-a sequence that helps enzymes get to their final destination
- Allosteric regulation: lac operon
- Covalent modification of enzyme: tyrosine kinase can activate or inactivate an enzyme by phosphorylating it.
How does compartmentation regulate enzyme activity?
Sequences in enzyme polypeptide chain target enzyme to ER, mitochondrion, nucleus because these organelles can read the signals on these proteins/chains
How does allosteric regulation reuglate enzyme activity?
A regulatory molecule (acting at a pocket distinct from the active site) changes the enzyme conformation to influence the active site and decrease (or in some cases, increase) enzyme activity.Controls the flux of material through a metabolic pathway
Give an example of allosteric regulation in organisms
E.coli usually grows on glucose, but if there is no glucose it can grow on lactose, the lactose causes certain enzymes to be switched on which can digest lactose.
How do covalent modifications regulate enzyme activity?
Change enzyme shape and activity-e.g. phosphorylation- tyrosine kinase can inactivate or activate an enzyme by phosphorylating it
How can feedback inhibition regulate enzyme activity?
·As product Z builds up, it…· inhibits A, this is done by allosteric inhibition it binds to the allosteric site on A.
Give an example of a reaction involving feedback inhibition
· Glycolysis has multiple control points including the early enzyme phosphofructokinase which is regulated by citrate, ADP and ATP.
Describe an example of an enzyme that uses allosteric regulation(3)
ATCase. The catalytic subunits are present as trimers in two hemispheres held together by regulatory subunits(R).
- The actives site is found on the inside of the hemisphere.
- With a lot of CTP, they can bind to the R subunits causing a change in the conformation of the enzyme so the two hemispheres are closed and there is no access to the active site.
What is ATCase?(3)
- Aspartate transcarbamoylase.
- ATCase forms part of the multi-enzyme complex CAD (alongside carbamoyl phosphate synthetase II and dihydroorotase)
- catalyses several steps in pyrimidine biosynthesis
Why do antibiotics target gyrase?(2)
- Gyrase catalyses ATP-dependent DNA supercoiling by a double- strand DNA break.
- Its sister enzyme Topo IV catalyses DNA relaxation by a similar mechanism
What do novobiotic and fluoroquinolones do?
Novobiotic- competitively inhibits the binding of ATP to gyrase Fluoroquinolones- inhibit DNA resealing by gyrase/topo IV. Introduces a double strand break into the DNA- this is reversible
Recall some enzyme related diseases(2)
• phenylketonuria: cannot convert Phe to Tyr. Buildup of Phe • glycogen storage disease- cannot mobilise glucose • Tay-Sachs disease-defect in processing a membrane ganglioside- neuronal damage and death
Recall some drug targets(3)
· Antibiotics : e.g. penicillins inhibit cell wall synthesis
· Anti-inflammatory agents: aspirin blocks prostaglandin
Anticancer drugs: methotrexate is a folate analogue: interferes with synthesis of DNA precursors
What is a perfect catalyst?
also called a diffusion limited enzyme is one that catalyses the reaction so well that the limiting factor is how quickly the substrate diffuses out of the active site. (kinetic perfection or catalytic perfection).
What is binding of enzyme and substrate dependent on?
The binding is not influenced by evolution as it depends on size, shape of enzyme and substrate.
How is evolution involved in enzymes?
· Overtime point mutations can speed up catalytic steps so that the chemistry is not the limiting factor but the binding of the enzyme and substrate.
How can we determine if reaction rate is affected by viscosity?(5)
- Enzyme and substrate in a reaction.
- Follow the rate of reaction.
- A small amount of substrate otherwise measuring the k3/ stickiness.
- The Km will be low because not a lot of substrate needs to be put in to get a high rate
- If the binding is limiting, then reaction rate is affected by viscosity so if
How does viscocity affect reaction rate?
The substrate will diffuse a lot less quickly when it is in a highly viscous solution
Recall the theoretical calculation for diffusion limited reaction
k3 /Km ~ 108 M-1s-1
§ Carbonic anhydrase: k3 is ~600,000 s-1 ; Km is 8 x 10-3. So appears to be diffusion limited § The turnover number is given by vmax/total concentration §
If you divide the 600,000 by the Km (carbonic anhydrase) is limited by the diffusion, it has evolved to the point where the chemistry is faster than the diffusion step.
If an enzyme after the calculation has 108M-1s-1 then it is diffusion limited
What is the gold-standard for determining if substrate binding is rate limiting?
- Determine complete free energy profile for the enzyme reaction
- Free energy of activation from all the small different steps
Describe an example of a perfect enzyme(4)
Triosephosphate isomerase is involved in glycolysis
- if you substitute protons with deuterium and tritium they slow the reaction down because they are heavier.
- It breaks the reaction down into a series of small steps which have much smaller free energy of activation,
- the substrate finds the A.S and makes the E-S complex and makes an intermediate which is converted into the product which is then released into the product.
Summarise the glycolysis process(3)
Glucose is a 6C molecule and pyruvate is a 3C molecule.
- The enzyme called aldolase (step 4) converts the substrate fructose 1-6 bisphosphate into 2 3C molecules, dihydroxyacetone phosphate and the other is called glyceraldehyde phosphate.
- Only glyceraldehyde 3 phosphate can be converted into pyruvate
Describe the nature of binding between substrate and perfect enzymes
Not bind too tightly or else it would slow the reaction
Name some classes of proteases
· proteases, serine, cysteine, aspartate groups and others use metal
Describe the structure of serine proteases
- It has a crevice and this where the substrate will be bound.
- Residue 195 is a serine which is very reactive and reacts with peptide bond and forms acyl enzyme intermediate and can be hydrolysed easily
3· All serine protease have active site serine and catalytic triad which is a constellation of active site groups making active site reactive
How are protease specificity determined?
· By the nature of the residue which is on N-terminal side of the peptide bond that will be cleaved.
Where do Chymotrypsin, Trypsin, and Elastase cleave?
Chymotrypsin will only cleave to peptide binds where the residue that are hydrophobic
Trypsin will cleave to negative charge
Elastase is on a narrow pocket
Describe the structure of elastase
· Elastase is constricted by two valine reside that project into binding pocket.
Describe the uncatalyzed hydrolysis of a peptide bond by water(3)
· We get the tetrahedral intermediate because carbon has 4 groups around it.
· Nitrogen picks up a proton in solution, the uncatalyzed has a high free energy activation.
- That is why proteins are stable because their free energy is so high to hydrolyse them
Draw the mechanism of uncatalysed hydrolysis reaction
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How do serine proteases catalyse hydrolysis?
· Serine proteases have very active serine which participates in reactions to form acyl intermediate and that binds to water.
Describe the serine protease hydrolysis mechanism(4)
· Instead of water binding, serine OH replaces water and the oxygen of OH attacks the carbonyl group of the peptide bond.
· Tetrahedral intermediate
· Break the peptide bond and release the carboxyl end. The other end is linked to the enzyme via the acyl link
· The intermediate is hydrolysed by water. Much easier to hydrolyse acyl compound than the peptide bond directly
· Results In release of serine-OH and the peptide chains (carboxyl group) of the peptide chain.
Why is an acyl intermediate necessary for hydrolysis of peptide bonds?
· Much easier to hydrolyse acyl compound than the peptide bond directly
Why is serine-OH so reactive? (5)
· Serine OH interacts with histidine and aspartate residue and form catalytic triad.
· Hydrogen bonding network between the three residues,
· The proton on serine-OH can be shifted onto the alkoxide group- charge relay
· Makes serine OH more negatively charge- more powerful nucleophile.
· All serine proteases have charge relay systems which potentiates the nucleophilicity of the serine involved in catalyses
Describe the movement of H+ in mitochondria(3)
· Protons are pumped from the inner mitochondrial membrane to the outside
· Inner mitochondrial membrane is highly impermeable so strong charge between the outside and the inside- proton motive force. More proton on the outside than inside
· Difference is an energy source used by rotary ATPases in the inner mitochondrial membrane. Use protons to synthesise ATP from ADP and phosphate.
How does ATPase rotary work?(6)
· Complex made of two parts: rota sitting in membrane which can turn as protons come outside mitochondria to inside.
· Rota is connected to spindle that protrudes through to the top part of the complex like a Stata stabilised by a protein link to the membrane
· So you have a rotating part and a stationary part.
· Electrostatic gradient provides energy.
· In the middle of the complex are three active sites which combine ADP and phosphate and a rota that rotates in 120 degree clicks which induces the synthesis of ATP
· Rotation cause ADP and phosphate to combine and expel water.
Describe the experiment used to visualise ATPase function(4)
· Rotary ATP synthase and attached to glass side
· Engineered the synthase so it was possible to attached to actin filament to the rotating spindle of the synthase. Actin filaments are long and can be stained and visualised
· Because enzymes are reversible, they were able to drive the rotation of spindle by the ATP being hydrolysed to ADP.
· Using microscope, you can see the movement of the spindle by the movement of the actin filament which moves in an arc in 120 degree clicks
Describe the mechanism of topoisomerase in untangling DNA(4)
· Topo is ATP driven clamp with a series of gates that can be opened and closed sequentially by the binding of and hydrolysis of ATP.
→The G-segment (gate segment) of the chromosome binds to the Topoisomerase II.
→ ATP is used to clamp the T-segment (target segment) of the chromosome.
→ The G-segment is broken, and the T-segment is pulled through.
→The G-segment is resealed and released via ATP hydrolysis.
How does Topo II separate DNA supercoiling?(2)
Uses ATP to be able to bind DNA segments from chromosome and pass through a double stranded break that It introduces in a second chromosome.
2. By crossing one DNA duplex to another, it can separate the strand
Summarise the action of actin and myosin as examples of enzyme machinery ATPase
The active site on actin is exposed to Ca2+ binds troponin
- The myosin head forms a cross-bridge with actin
The enzyme at the binding site on myosin is called ATPase. - The energy released during ATP hydrolysis changes the angle of the myosin head into a “cocked” position.
- During the power stroke, the myosin head bends and ADP and phosphate are released
- A new molecule of ATP attaches to the myosin head causing the cross-bridge to detach ATP hydrolyses to ADP and phosphate, which returns the myosin to the cocked position
what is the turnover number?
the maximum number of chemical conversions of substrate molecules per second that a catalytic site will execute for a given enzyme concentration
what are properties of allosteric enzymes?(5)
→multi subunit complexes
→regulatory sites and catalytic sites are on different subunits
→regulation happens via conformational changes
→involved in feedback inhibition
→non michaelis menten kinetics
how does ATP synthase work? (6)
→ATP Catalysis begins when protons pass through the part of the enzyme that lies in the cell membrane, causing it to turn.
→The central core then rotates inside the top half of the enzyme.
→this region holds an ATP molecule and pulls in ADP and an inorganic phosphate group in the neighbouring subunit.
→As the core rotates, the subunit with ATP loosens, and the section holding ADP closes.
→The original ATP molecule is released, and a new one is formed from the ADP.
→ The cycle repeats.
what is the catalytic triad?
→Ser 195 may get its reactivity due to its positioning next to His 57 and Asp 102.
→This is a catalytic triad, which makes the serine much more electronegative.
→ triad is found in all proteases.
how does trypsin work?
→cleaves after Lys, Arg (which are positively charged), due to its negative pocket/cleft
how does chymotrypsin work?
→cleaves after Phe, Trp, Tyr (which are aromatic and hydrophobic) due to its hydrophobic pocket/cleft
how do perfect enzymes work?(3)
→ instead of there being a big energy change and the reaction happening by conversion of A to Z
→it happens via intermediates that don’t have a high energy change between them, making the conversion easier.
→The reaction is limited by E + S (substrate diffusion into the enzyme), so decreasing the energy levels doesn’t increase the efficiency/rate of reaction.
