Module 2.4 - Enzymes Flashcards
(MA) General points about enzymes.
- enzymes are globular proteins
- enzymes are specific
- refer to active site if talking about how enzymes work/denature/anything binding to them/function
- substrates are complementary in shape to the ACTIVE SITE of the enzyme
- talking about the enzyme substrate complexes (ESCs) forming/not forming
- lock + key hypothesis/induced fit hypothesis when talking about enzyme action
(MA) Why are enzymes knew as specific?
- shape of active site
- complementary
- to correct substrate
- forms ESCs
- any other substrate will not
(MA) Describe enzyme action using the induced fit hypothesis as an example.
- as the substrate binds to the active site, the enzyme changes shape slightly
- active site is tighter around substrate molecules
- oppositely charged groups on the substrate + active site interact + hold the substrate molecule in place. This is the enzyme substrate complex (ESC)
- enzyme’s shape change puts strain on the bonds in the substrate which destabilises it, causing the reaction to occur more easily
- the product(s) is formed (enzyme product complex) + because it’s a different shape to the reactant it’s released from the active site
(MA) Explain the effect of temperature on enzyme activity.
Up to optimum:
-as molecules are heated they gain KE + move faster resulting in more frequent collisions
-this results in more ESCs + therefore a higher (max) rate of reaction + more product formed
Above optimum:
-molecules have more KE
-enzymes vibrate too much + weaker bonds are broken (ionic + H)
-tertiary structure of the enzymes are changed
-meaning active site loses its complementary shape
-no ESCs can form as substrate doesn’t fit into active site
-enzymes have denatures
-this is irreversible so reaction stops
(MA) Explain the effect of pH on enzyme activity.
Not at optimum:
-change in pH or H+ ions alters distribution of charge on enzyme molecule
-causes H bonds + ionic bonds to break
-means the enzyme loses its tertiary structure
-changes the shape of the active site of the enzyme
-substrates are no longer attracted to the active site because H+ ions have altered its charge
-substrates cant bind to active site as its no longer complementary
-no ESCs can form = no product = no reaction
-enzymes are denatured at extremes of pH (for that enzyme)
At optimum:
-at optimum, the conc of H+ ions gives the tertiary structure the best shape = most complementary active site
(MA) Explain the effect of increasing enzyme concentration on enzyme activity when there is a fixed concentration of substrate.
-no enzyme = no ESCs = no reaction
As long as the substrate is in excess:
-as enzyme conc increases, the rate of reaction increases
-more enzymes = more active sites = more ESCs form so more product = higher rate of reaction
-as long as substrate in excess the rate will continue to rise w an increase in enzyme conc
As substrate is used up (not in excess):
-all substrate molecules are occupying active sites; max rate of reaction
-increasing enzyme conc further will have no effect on rate
-when substrate is used up the rate will decrease as less product is formed
-substrate is limiting
(MA) Explain the effect of substrate concentration on enzyme activity when there’s a fixed concentration of enzyme.
-no substrate = no ESCs = no reaction
As long as enzyme is in excess:
-as substrate conc increases, rate of reaction increases
-more substrate = more frequent collisions between active sites + substrate = more ESCs = more product = higher rate
When all active sites occupied:
-it’s not possible for more ESCs to form at any one time = increasing the substrate conc further has no effect of rate, rate plateaus
-enzyme conc is a limiting factor
(MA) How do competitive inhibitors work?
- competitive inhibitors have a similar shape to an enzymes’ substrate
- their shape is complementary to the active site so they can bind with it + block it, forming an EIC
- prevents ESCs from forming + slows rate of reaction so no products can be formed
- don’t bind permanently to active site; is reversible
(MA) How do non-competitive inhibitors work?
- fit into the allosteric site on an enzyme
- alters the tertiary structure of the enzyme + changes the shape of the active site
- meaning the substrate can no longer fit into the active site, so no ESCs can form; rate of reaction decreases
- bind permanently to the enzymes; their effect is reversible; the enzymes become useless
(MA) Explain the effect of competitive inhibitor/substrate concentration on enzyme activity.
- rate determined by relative concentrations of substrate/inhibitor
- more inhibition if substrate conc is lower than inhibitor conc
- there’s a higher chance of inhibitor entering active site than substrate = less ESCs = less product
- more substrate = less inhibition = higher rate of reaction as more likely to collide w active site than an inhibitor
- effects can be reversed by increasing substrate conc
(MA) Explain the effect of non-competitive inhibitors on enzyme activity.
- increasing substrate conc has no effect on the rate because they bind irreversibly, if all enzymes have a non competitive inhibitor bound, the reaction will stop
- changing the conc of inhibitors will reduce the rate further = fewer ESCs = less product
Why is the tertiary structure of enzymes important?
Gives the specific shape of the active site so that it is complementary to the substrate
Give examples of what enzymes might be used for.
- control metabolic reactions
- control DNA replication
- protein synthesis
- formation/breakage of bonds
Which enzyme breaks down lactose?
Lactase
Which enzyme breaks down ATP?
ATPase
Why do the enzymes in endotherms usually not need to be adapted for extremes?
- maintain internal body temp
- maintain optimum temps for thermoregulation
How do heterotrophic feeders use enzymes in digestion?
- consume other organisms + digest them in the body w the aid of enzymes
- e.g. animals
How do saprophytic feeders use enzymes in digestion?
- release enzymes onto their food
- digests the food outside of the body
- absorb the monomers
- e.g. fungi
How can enzymes be used as a defence again disease?
Digest or help digest pathogens
What kind of enzymes are extracellular enzymes?
Most digestive enzymes
Where are intracellular enzymes found?
Catalyse within cytoplasm/on the membrane
What do you call a cofactors permanently bound to an enzyme and through what type of bond does this occur?
- prosthetic group
- covalent bond
Give an example of a prosthetic group.
The carbonic anhydrase enzyme contains a zinc ion permanently bound as the prosthetic group
How can cofactors act as co-substrates?
They + the substrate together form the correct shape to bind to the active site of the enzyme
Why do cofactors alter the charge distribution on the surface of the active site of the enzyme or on the surface of the substrate?
Make the temporary bond in the ESC easier to form
What are coenzymes?
- small organic non protein molecules
- bind temporarily to the active site of the enzyme molecule
- coenzyme is chemically changed during the reaction
- need to be recycled to their original state, sometimes by a different enzyme
What is the pattern in the collisions between enzymes and substrates?
There’s no pattern! They’re random!!
What is the optimum temperature?
The temp that gives the max rate of reaction. Balance between max collision + the vibration of the enzyme molecules
What does pH measure?
H+ ions
If there are more H+ ions, how does this affect the pH?
More acidic
Why can the distribution of H+ ions affect the enzyme’s ability to bind the the substrate?
Large numbers of H+ ions attract -vely charged parts of the enzyme + repel positively charged parts. Interferes w the H bonds + ionic bonds giving the enzyme its tertiary structure + therefore the specific active site
What are enzyme inhibitors?
Molecules that slow down the rate of an enzyme controlled reaction by affecting the enzyme molecule
What are the two types of enzyme inhibitors?
- competitive
- non competitive
What is a catalyst?
Chemical that speeds up the rate of reaction but remains unchanged and reusable at the end of the reaction. Small amount of catalyst can catalyse the conversion of a large number of substrate molecules into product molecules
What does the term turnover number describe?
the number of reactions an enzyme molecule can catalyse per second
Why is the tertiary structure of the active site crucial?
Complementary to the shape of the substrate
What does it mean if a metabolic pathway is described as catabolic?
metabolites are broken down to smaller molecules + release energy
What does it mean if a metabolic pathway is described as anabolic?
Energy is used to synthesise larger molecules from smaller ones
Give some examples of complex metabolic pathways that involve enzymes.
- respiration
- photosynthesis
Describe the role and structure of the catalase enzyme (intracellular).
- found in nearly all living organisms exposed to oxygen
- protects cells from damage by reactive oxygen by quickly breaking down reactive hydrogen peroxide (harmful by-product of metabolic reactions) to H2O + O2
- consists of 4 polypeptide chains + contains a haem group w iron
- fastest acting enzyme having the highest turnover no. (6 million per second)
- eukaryotes: catalase found inside small vesicles called peroxisomes
- when WBCs ingest pathogens they use catalase help kill invading microbes
- optimum pH (humans): 7
- optimum temp (humans): 45C
Describe the role of amylase enzymes (extracellular).
- produced in salivary glands
- acts in mouth to digest polysaccharide starch to disaccharide maltose
- made in pancreas + acts to catalyse the same reaction in the lumen of the small intestine
Describe the role of trypsin enzymes (extracellular).
- made in pancreas
- acts in lumen of small intestine to digest proteins into smaller peptides by hydrolysing peptide bonds
- optimum pH: between 7.5 & 8.5
What is the term to describe a cofactor that is permanently bound by covalent bonds to an enzyme molecule?
prosthetic group
What is a cofactor?
a substance that has to be present to ensure an enzyme-catalysed reaction takes place at appropriate rate. Some cofactors (prosthetic groups) are part of the enzyme structure, and others (mineral ion cofactors + organic coenzymes) form temporary associations with the enzyme
What is the prosthetic group in carbonic anhydrase and what is carbonic anhydrase’s role?
- contains zinc ions permanently bound as a prosthetic group to its active site
- found in erythrocytes + catalyses interconversion of CO2 + water to H2CO3, which breaks down to protons + HCO3- ions
- reaction may happen in either direction depending on conc of substrate/product molecules
- CO2 + H2O H2CO3 H+ + HCO3-
Do cofactor ions have to be permanently bound to the enzyme?
not necessarily, dependent on the enzyme
How can some cofactors act as co-substrates?
they and the substrate together form the correct shape to bind to the active site of the enzyme
How can cofactors work by making the bonds between substrate and enzyme easier to form?
- change the charge distribution on the surface of the substrate molecule/enzyme’s active site
- make temporary bonds in ESC easier to form
Give one enzyme that requires an ion not permanently bound to the enzyme to work
Amylase enzyme (that digests starch into maltose) requires Cl- ions to work
What are coenzymes?
- small organic non protein molecules
- bind temporarily to active site of enzyme molecules
- either just before/at same time as substrate binds
- coenzymes are chemically changed in the reaction + need to be recycled to their original state, sometimes by other enzymes
What coenzyme is derived from the B12 vitamin and what human deficiency disease can come as a result of not enough B12?
- cobalamin coenzymes
- pernicious anaemia (progressive + fatal anaemia)
What coenzyme is derived from folic acid vitamins and what human deficiency disease can come as a result of not enough folic acid?
- tetrahydrofolate
- megablastic anaemia (large, irregularly shaped erythrocytes)
What coenzyme is derived from nicotinamide, B3 vitamins and what human deficiency disease can come as a result of not enough B3?
- NAD, NADP
- pellagra (diarrhoea, dermatitis + dementia)
What coenzyme is derived from the pantothenate, B6 vitamin and what human deficiency disease can come as a result of not enough B6?
- coenzyme A
- elevated blood-plasma triglyceride levels
What coenzyme is derived from the thiamine, B1 vitamin and what human deficiency disease can come as a result of not enough B1?
- thiamine pyrophosphate
- beriberi (mental confusion, irregular heartbeat, muscular weakness, paralysis + heart failure)
Describe the lock and key hypothesis.
- enzyme’s active site is complementary in shape to substrate molecule
- substrate (key) can fit into the enzyme’s active site (lock)
How does an enzyme work, using the lock and key hypothesis?
- substrate molecules + enzymes molecules have KE + constantly move randomly
- if substrate molecules successfully collide w an enzyme molecule an ESC forms as substrate molecule fits into complementary shaped active site on enzyme molecule
- substrate molecules broken down/built up into produce molecule(s) + form EPC (enzyme product complex) whilst still in active site
- product molecules leave active site
- enzyme molecule now able to form another ESC
- small no. of enzyme molecules can therefore convert a large no. of substrate molecules into product molecules
What does the lock and key hypothesis not explain that the induced fit hypothesis does?
how the enzyme substrate complex is stabilised
How does an enzyme work, using the induced fit hypothesis?
-substrate molecules fit into enzyme’s active site changing its shape slightly to mould around substrate
-enzyme’s active site has complementary shape to substrate molecule but on binding the subtle changes of shape of R groups of amino acids making up active site give a more precise conformation that exactly fits the substrate
-moulding means substrate binds more effectively to active site
-ESC is formed + no covalent forces e.g. H bonds, ionic bonds, London forces + hydrophobic interactions bind substrate to enzyme’s active site
-when substrate converted to product they’re still in active site: product-substrate complex
-as product molecules have slightly different shape from substrate they detach from active site
-enzyme molecule now free to catalyse another reaction w another substrate of the same type
E + S –> ESC –> EPC –> E + P
How do enzymes work in terms of energy?
- chemical reactions need energy to activate them, often in form of heat as increases KE of molecules so move about more + more likely to collide successfully + react
- living cell: temp can’t be raised too much or proteins denature + lipids melt
- as enzyme’s active site specific to only the substrate molecules they bring substrate molecules close enough together to react w/out the need for excessive heat
- lower activation energy + hence speed up metabolic reactions
What happens to a substance when it is heated?
- extra energy in form of heat means molecules move faster
- increases rate of collisions between molecules
- increases force w which they collide as moving faster
How does heat affect the rate of formation of an enzyme substrate complex up to the optimum and why?
- both types of molecule will gain KE and move faster
- increases rate (number per second) of successful collisions
- therefore rate of formation of ESCs increases + RoR increases, increasing the number of EPCs per second up to a point
- enzyme has an optimum temp at which RoR is at its fastest
Explain how an enzyme becomes denatured through high temperatures.
- breaks some of the weak bonds e.g. H/ionic bonds, holding tertiary structure of enzyme’s active site together
- as active site begins to change shape substrate molecules won’t fit so well + RoR begins to decrease
- as more heat applied, shape of enzyme’s active site no longer complementary to shape of substrate molecule
- reaction can’t proceed at all
- enzyme is dentatured
What is the optimum temperature?
- temp at which the enzyme works best
- temp at which the enzyme has its max RoR
Why do some enzymes have a different optimum temperature?
- organisms are adapted to living in different temperatures
- psychrophilic bacteria adapted to cold conditions; low optimum temp
- thermophilic bacteria adapted to hot conditions of hot springs; high optimum temp (heat stable)
How would an enzyme be adapted to high temperatures?
-more disulfide bridges as don’t break at high temps + keep molecule shape stable
How do you calculate RoR?
1/(time taken to reach end point)
What is the unit for RoR?
s^-1
What is the temperature coefficient, Q10?
Refers to the increase in the rate of. Process when the temperature is increased by 10°C
What is the equations for Q10?
(Rate of reaction at T+10 °C)/(rate of reaction at T°C)
What is Q10 of reactions in a test tube and what does this mean?
2, RoR doubles for each 10°C increase
What does HCl dissociate to?
H+ & Cl-
What is a buffer?
Something that resists changes in pH
How do buffers work?
Donate or accept H+ ions
How does haemoglobin act as a buffer?
Accepts or donates protons to resist pH changes
What does sulfuric acid dissociate into?
H+ & HSO4 -
What does lactic acid dissociate into?
H+ and lactate
What does pyruvic acid dissociate into?
H+ and pyruvate
How is pH worked out?
log 1/[H+]
How does a change in pH affect the bonds within molecules?
- proton has a positive charges so attracted to negative ions/molecules/parts
- excess H+ ions interfere w H bonds & ionic bonds holding together tertiary structure so active site of enzyme changes shape. If substrate doesn’t fit into active site RoR will be lowered
- increasing [H+] alters charges on active site of enzyme molecules as more protons cluster around negatively charged groups in active site. Interferes w binding of substrate to active site
Describe what is meant by enzymes having a narrow range of pH.
- small changes in pH either side of optimum slow RoR as shape of active site is disrupted
- is normal pH restored H bonds can reform + active site’s shape is restored
- extremes of pH: enzyme’s active site may be permanently changed. When enzyme is denatured it can’t catalyse reactions
What is the optimum pH for intracellular enzymes?
7
Give examples of extracellular enzymes that don’t have a pH of 7.
- amylase to digest starch to maltose: pH 6.8
- pepsin in HCl: pH 1-2 (digests proteins into smaller polypeptides)
- small intestine salts in bile made in liver neutralise it + raise pH to around 7.8. Optimum for protein digesting enzymes (trypsin + enterokinase) to further digest peptides into amino acids
What is the effect of changing the substrate concentration on the RoR?
-no substrate = no enzyme catalysed reaction as no substrate molecules to fit into active sites of enzymes so no ESCs can form
-as conc increases ROR increases:
>more ESCs can form
>more product molecules form
>substrate conc is limiting reaction as it increases RoR increases
>substrate conc is the limiting factor
-as conc increases further reaction will reach max rate:
>adding more substrate won’t increase RoR
>all enzyme’s active sites occupied w substrate molecules
>if more substrate added they can’t successfully collide w + fit into an enzyme’s active site
In cells, what does the enzyme concentration/availability depend on?
Rate of synthesis of the enzyme + its degradation
How can enzyme synthesis affect the concentration/availability of enzymes?
depending on cell’s needs, genes for synthesising particular proteins can be switched on/off
What is enzyme degradation?
-protein component of living cells constantly being turned over. Cells continuously degrading old enzyme molecules to their component amino acids + synthesising new enzyme molecules from the amino acids
What are the advantages of enzyme degradation?
- elimination of abnormally shaped proteins that might otherwise accumulate + harm cell
- regulation of metabolism in the cell by eliminating any superfluous (surplus to requirements) enzymes
How does increasing the enzyme concentration in an enzyme controlled reaction affect the RoR?
-more active sites become available
-more successful collisions between enzyme + substrate
-more ESCs form per unit time so RoR increases
-enzyme conc is limiting factor
If Substrate Conc Is Fixed Or Limited:
-enzyme conc increasing doesn’t increase RoR as active sites of extra enzyme molecules won’t be occupied by substrate molecules
-enzyme conc isn’t limiting factor
-substrate conc is limiting factor as preventing RoR from increasing
When is the rate of reaction between reactants fastest?
At the start
How does the rate of reaction change over the course of a reaction?
- beginning of reaction: great chance of substrate molecules colliding successfully w enzyme’s active site
- as proceeds: substrate molecules are used up as they’re converted to product molecules, so substrate conc decreases
- so frequency of successful collisions between enzyme + substrate decreases as some enzymes may collide w product so RoR slows
- initial RoR give the max RoR for an enzyme under a particular experimental condition
What is an inhibitor?
A substance that reduces or stops a reaction by reducing activity of an enzyme
How do competitive inhibitors work?
- competitive inhibitor fits into active site + so substrate molecules can’t enter
- amount of inhibition depends on relative concs of substrate + inhibitor. More inhibitors = more inhibitors colliding w active sites so greater effect
- increasing substrate conc effectively dilutes inhibitor effect. If enough substrate added, inhibitor unlikely to collide w an enzyme
- compete w substrate molecules for position on enzyme’s active site, forming an enzyme-inhibitor complex (catalytically inactive)
- once on active site, inhibitor isn’t changed by enzyme like the substrate would be
- inhibitor’s presence prevents substrate molecule joining to active site reducing rate of ESC formation + product molecule formation
- most enzyme inhibition by competitive inhibitors is reversible
- if competitive inhibitors binds irreversibly to enzyme’s active site its called an inactivator
What do you call a competitive inhibitor that binds irreversibly to the active site of an enzyme?
Inactivator
Describe the graph for the substrate conc against RoR with a competitive inhibitor.
Slower rate of reaction up to max. Straight line up and higher substrate conc needed for max RoR
How do non-competitive inhibitors work?
- attach to allosteric site of an enzyme molecule away from active site disrupting enzyme’s tertiary structure + changes its shape
- changes shape of active site so not complementary to substrate molecule + substrate can’t bind to active site. ESCs can’t form
- max RoR reduced. Increasing substrate conc might reach new max faster, but can’t reach uninhibited max
- more inhibitors = greater degree of inhibition as more enzyme molecules distorted + can’t form ESCs/complete catalytic reaction involving ESCs
- some bind reversibly to allosteric site but others bind irreversibly
How does end product inhibition work?
- product molecules of reaction stay tightly bound to enzyme
- enzyme can’t form any more product
- an example of negative feedback
How can a series of enzyme catalysed reactions be used to control metabolic sequences such as photosynthesis and respiration?
- product of one enzyme catalysed reaction is the substrate for the next in the metabolic pathway
- cells don’t need to accumulate too much of end product so product of last enzyme catalysed reaction in metabolic pathway may attach to part of first enzyme in pathway but not its active site
- binding changes shape of first enzyme’s active site preventing pathway from running. Non competitive inhibition but reversible
- when conc of product within cell falls those molecules detach from first enzyme + allow active site to resume normal shape: metabolic pathway can run again
Why do multi-enzyme complexes increase the efficiency of metabolic reactions even though they don’t increase the substrate concentration?
Keep same enzyme + substrate molecules in same vicinity + reduce diffusion time
Why are metabolic reactions often carried out in particular regions/organelles of the cell?
increase efficiency of metabolism (some enzyme within organelles bound to the organelles membrane)
How does cyanide work?
- potassium cyanide (KCN) is highly toxic as inhibits aerobic respiration (as well as catalase)
- when ingested KCN is hydrolysed to produce hydrogen cyanide, toxic gas that can readily dissociate into H+ and CN- ions
- CN- ions binds irreversibly to an enzyme in mitochondria + inhibit final stage of aerobic respiration, so earlier stages can’t run + aerobic respiration stops
How does snake venom work?
- venom of green mamba snake has a chemical that inhibits enzyme acetylcholinesterase (AChE)
- enzyme important for neuromuscular synapses to break down neurotransmitter (acetylcholine, ACh)
- if inhibited, ACh stays attached to receptors on muscle membrane + keeps muscle contracted
- causes paralysis as movement depends on muscles being able to contract + relax alternately. If muscles involved in breathing are paralysed victims die from suffocation
How does aspirin work as a medicinal drug acting by enzyme inhibition?
- 1971: Vane discovered salicyclic acid binds to enzymes that catalyse formation of prostaglandins
- prevents its formation. They’re cell signalling molecules produced by cells when tissues infected/damaged
- prostaglandins make nerve cells more sensitive to pain + increase swelling during inflammation
- aspirin can reduce chance of blood clots in blood vessels (many take low dose to reduce risk of stroke)
- aspirin can damage stomach lining in children under 12
How does ATPase work as a medicinal drug acting by enzyme inhibition?
- extracts from purple foxglove leaves used to treat heart failure + atrial arrhythmia (abnormal beat rate of atria)
- chemicals identified as cardiac glycosides (digitalis, digitoxin. digitalin, digoxin)
- inhibit the sodium potassium pump in cell membrane of heart cell muscles + allow more Ca ions to enter the cells
- Ca ions increase muscle contraction which strengthens heartbeat
How do ACE inhibitors as a medicinal drug acting by enzyme inhibition?
- inhibits angiotensin converting enzyme (ACE) which operates a metabolic pathway to increase blood pressure
- lower blood pressure in patients with hypertension who can’t take beta-blockers
- treat heart failure, low dose first then blood pressure checked in case it falls too low
- minimise risk of second heart attack/stroke in patients who’ve suffered a myocardial infarction
How do protease inhibitors work as a medicinal drug by enzyme inhibition?
- e.g. amprenavir, ritonavir
- treat viral infections
- prevent replication of virus particles within host cells by inhibiting protease enzymes so they viral coats can’t be made
- often inhibit viral protease enzymes by competitive inhibition
How do nucleoside reverse transcriptase inhibitors work as a medicinal drug through enzyme inhibition?
- many antiviral drugs (e.g. zidovudine + abacavir) to treat HIV positive patients are nucleoside reverse transcriptase inhibitors
- inhibit enzymes involved in making DNA using viral RNA as a template
