2.1.4 Enzymes

Question 1

what are enzymes also known as and why

Answer 1

biological catalysts

• a catalyst is a substance that speeds up the rate a chemical reaction and remains unchanged and unused
• bio catalysts are found in living organisms and they catalyse metabolic reactions (reactions that occur in living cells)

Question 2

how do enzymes affect metabolism at a cellular level e.g respiration

Answer 2

intracellular (working within cells) enzymes affect metabolism at a cellular level

• e.g catalase is an enzyme that works inside cells to catalyse breakdown of hydrogen peroxide to oxygen + water
• important bc hydrogen peroxide is a toxic by product of cellular reactions - if build up can kill cells
• catalyse consists of 4 polypeptide chains and a haem group w iron
• found in peroxisomes
• optimum pH is 7 ,opt temp is 45degrees (inhumans)

Question 3

how do enzymes affect metabolism at a whole organism level e.g. digestion in mammals

Answer 3

extracellular (working outside of cells) enzymes affect metabolism at a whole organism level

• e.g. amylase and trypsin work outside cells in human digestive system
• amylase secreted by salivary glands + found in saliva catalyses the hydrolysis of starch into maltose in mouth
• amylase is also made in pancreas to catalyse same reaction in lumen of small intestine
• trypsin catalyses the hydrolysis of peptide bonds (turning big polypeptides into smaller ones) this is produced by the pancreas + secreted into lumen of small intestine (opt pH 7.5-8.5)

Question 4

Describe how a metabolic pathway works and what things are involved in it

Answer 4

• each metabolic pathway in a living cell is a series of consecutive reactions
• each step catalysed by a specific enzyme to make a specific product
• reactants and intermediates act as substrates for specific enzymes
• reactants/intermediates/products are known as metabolites
• in catabolic metabolic pathways, metabolites r broken down into smaller molecules + release energy
• in anabolic metabolic pathways, energy is used to synthesise larger molecules from smaller ones
• respiration/photosynthesis r examples of metabolic pathways

Question 5

what is the structure of an enzyme

Answer 5

• enzymes r globular proteins
• they have an active site
• this active site is part of the enzyme where is substrate molecule (substance that enzymes interacts w) binds to
• this has a specific shape which is determined by the enzymes tertiary structure
• the substrate molecule is complementary to the active site of an enzyme
• when they bind it is known as an enzyme-substrate complex
• therefore each enzyme is highly specific to its function in that it can only catalyse a reaction involving a particular type of substrate molecule
• the shape of the AS can be altered by changed in temp and pH - affecting bonds in proteins in tertiary struc

Question 6

how does the structure of an enzyme affect its function

Answer 6

• the struc enables them carry out functions (AcSite)
• instructions for making enzymes r in genes
• if there is a mutation this might alter the primary sequence and hence the enzymes tertiary struc + stop it from functioning
• also catalyse formation of organisms structural components e.g. collagen in bone/cartilage/blood vessel walls
• sometimes enzymes need cofactors to work

Question 7

how do enzymes speed up reactions?

Answer 7

in every reaction a certain amount of activation energy(min energy needed usually heat) is needed to start it
- the heat increases kinetic energy so they move more and r more likely to successfully collide w each other to react
- enzymes reduce the activation energy needed by the reaction hence speeding up metabolic reactions
this happens when a enzyme-substrate complex is formed bc:
1. enzyme can hold 2 substrate molecule closer together to reduce an repulsion between them so they bond more easily
2. in catalysing a breakdown reac - purring substrate in active site puts strain on bonds in the substrate so more likely to breakdown

Question 8

what r the 2 models of enzyme actions

Answer 8

1. lock and key hypothesis

2. the induced fit hypothesis

Question 9

what is the lock and key hypothesis and draw diagram

Answer 9

• where substrate fits into the active site of an enzyme like how a lock fits into a key - they are complementary in shape
  1. substrate molecule + enzymes molecules have kinetic energy and move randomly
  2. if substrate collides successfully w enzyme ES complexed r formed (complementary shapes)
  3. substrates broken down or built up into product molecules forming EP complexes while in active site
  4. product leaves AS
  5. enzyme can repeat process again

(((((1. substrate molecule fits into enzymes active site - temp H bonds hold them together to form an -enzyme-substrate complex

2. enzymes catalyses reaction to form an enzyme-product complex (either get broken down or synthesised)
3. products leave enzyme))))) - diagram annotation

diagram

Question 10

what is the induced fit hypothesis (diagram)

Answer 10

• lock and key model does not explain how ES complexes r stabilised
• 1959 Daniel Koshland modified L/K model
• suggested that active site is not a fixed rigid structure but at the presence of a substrate molecule it induces a shape change in the active site so they fit well
  1. active site is still complementary to shape of substrate molecule
  2. but on binding the shape of R groups of amino acids in AS change slightly to fit the substrate molecule more precisely and effectively
  3. ES complexed r formed AND non covalent bonds e.g. Hbonds/ionic attractions also help hold it in place
  4. EP complex forms
  5. product molecules have diff shape to substrate hence leave active site of enzyme
  6. enzyme repeats process

Question 11

what is a cofactor

Answer 11

a non protein substance that is bound to an enzyme to help it catalyse reactions

Question 12

what types of cofactors are there

Answer 12

1. prosthetic groups
   - these are tightly bound to the enzyme by covalent bonds and part of its structure
2. inorganic cofactors (not containing carbon)
   - help binds AS to substrate for from ES complexes hence increasing rate of reaction
   - dont directly participate in reaction so dont get changed
   - some act as co-substrates: they + substrate together form correct shape to bind to AS
   - some change the charge dist of surface of substrate or active site to make the temp bonds in ES complex easier to form
3. organic cofactors aka COENZYMES
   - participate in the reaction and r changed by it
   - act as carriers to move chemical groups between diff enzymes
   - recycled in the process

Question 13

give examples of these types of cofactors

Answer 13

1. prosthetic group:
   example: Zinc (Zn2+) is a prosthetic group for carbonic anhydrase (enzyme in read blood cells), which catalyses production of carbonic acid from water and carbon dioxide - they r a permanent part of AS
2. inorganic cofactor:
   e. g. Chloride ions (Cl-) are inorganic cofactors 4 the enzyme amylase to break down starch to maltose
3. coenzymes:
   e. g. vitamins r sources of coenzymes
   - coenzyme NAD is derived from vit B3
   - coenzyme A derived from B6
   - coenzyme tetrahydrofolate derived from folic acid

Question 14

what factors affect enzyme activity

Answer 14

1. temp
2. pH
3. enzyme conc
4. substrate conc

Question 15

how does temp affect enzyme activity

Answer 15

• the optimum temp (for an enzyme) is the temp at which the rate of reaction is maximum and the enzyme works best
  what happens below and at optimum temp:
  1. if mixture if heated both substrate molecules and enzyme will gain kinetic energy and move faster
  2. increases rate of successful collisions
  3. rate of formation of ES complexes increase + rate of formation of EP complexes increased
  4. hence rate of reaction increases
  what happens above optimum temp:
  1. molecules will vibrate
  2. weak bonds e.g H bonds + ionic bonds in tertiary struc of enzyme might break
  3. active site shape changes irreversibly
  4. substrate molecule r not longer complementary to active site (less ES complexes and EP complexes)
  5. rate of reaction decreases
  6. then reaction cannot proceed and the enzyme is DENATURED!!!

opt temp humans: 37 - for organisms in cold environs e.g. psychrophilic bacteria - opt temp is low temp
or thermophilic bac -enzymes work at high temps bc it lives in hot springs (enzyme has loads of disulfide bonds)

Question 16

draw the shape of the a graph of the effect of temp on enzyme activity

Answer 16

graph

Question 17

what is the temperature coefficient (Q10)

Answer 17

(Q10) value or temperature coefficient for a reaction shows how much the rate of reaction changes when the temp is raised by 10 °C
Equation:

Q10= rate of reaction at (T+10)°C/rate of reaction at T°C)

for most reactions the temperature coefficient is 2 meaning the rate of reaction doubles when the temp of the reaction is raised by 10°C - sometimes its 3
when the temp is above opt temp the Q10 value decreases as the enzyme deantures

Question 18

what is pH

Answer 18

indicates the how acidic/alkaline/neutral a sub is

0-6 acidic 7 is neutral and 8-14 is alkaline
H+ ions make a solution more acidic 
OH- ions make a solution more alkaline
pH is worked out : log(1/H+) 
hydrogen ions r protons

Question 19

what is a buffer

Answer 19

buffer is something that resists changes in pH

Question 20

how does a buffer work and give examples of when it is used/occurs

Answer 20

e. g. chemicals in blood such or proteins such as haemoglobin can donate or accept H+ ions to keep the blood pH close to 7.4 and stable - they act as buffers
- in the lab buffer solutions r used to maintain desire pH or keep pH stable

Question 21

how does changes in pH affect the enzymes active site

Answer 21

• H+ ions or protons r attracted to negatively charged ions/molecules etc
• excess H+ ions will interfere w H bonds and Ionic bonds in the secondary/tertiary structure of an enzyme
• they may alter charges on the active site of enzymes molecules as more protons will cluster around negatively charged R groups in amino acids
• they may cause the enzyme to change shape and hence may no longer b complementary to the substrate
• hence rate of reaction might slow down

Question 22

how does pH affect the rate of enzyme activity

Answer 22

enzymes work in a narrow range of pH:

• small changes, reducing or increasing pH away from optimum pH reduces rate of reaction bc conc of H+ in solution affect tertiary struc of enzyme
• hence shape of active site is disrupted
• if normal opt pH is restored H+ can reform and active site shape is restored
• at extremes of pH enzymes active site is permanently changed and denatured hence no reaction occurs

Question 23

draw shape of graph of effect of pH on enzyme activity

Answer 23

graph

Question 24

what is the opt pH for enzymes?

Answer 24

enzymes working intracellularly have pH close to 7

• enzymes working extracellularly differ :
  1. amylase enzymes digest starch to maltose work best at 6.8
  2. HCl acid in stomach means that protease enzyme pepsin works optimally at pH 1/2
  3. salts in bile made in liver neutralise small intestine - trypsin works best at 7.8 - catalyses breakdown of peptides to amino acids

Question 25

define concentration

Answer 25

the number of molecules per unit volume

Question 26

what is a limiting factor

Answer 26

a variable that slows down the rate of a reaction

Question 27

what is the effect of substrate conc on rate of enzyme controlled reactions

Answer 27

no substrate present = no collisions = no ES complexes = no reaction

as conc of substrate increases, the rate of reaction increases to a certain point bc:

1. as it increases more ES complexes form
2. more EP complexes form and more products form
3. substrate conc is the limiting factor bc it is limiting the reaction - the more u increase the more reaction increases
4. as the conc increases reaction will reach its max rate
5. increasing conc of substrates will not increase reaction further aka the rate of reaction will stay the same
6. it is no longer the limiting factor
7. this is bc enzymes active sites r occupied and if more subs r added it will not collide w the occupied active sites
8. enzyme conc is now liming factor

Question 28

draw shape of graph of substrate conc of rate of enzyme activity

Answer 28

graph

Question 29

what affects the availability of enzymes in cells

Answer 29

• depends on the rte of synthesis of enzyme and its rate of degradation
• enzyme synthesis depends on whether or not genes for synthesising enzymes r switched on or off

Question 30

what r the advantages of enzyme degradation

Answer 30

cells r continuously degrading old enzyme molecules into their component amino acids and synthesising new enzyme molecules from amino acids

1. it eliminates abnormal enzymes that might accumulate and harm the cell
2. it regulates metabolism in cell by eliminating extra enzymes

Question 31

how does enzyme conc affect enzyme acitivity

Answer 31

as enzyme conc increases:
1. more active sites on enzyme become available
more successful collisions occur between enzyme and substrate molecules
2. more ES complexed and more EP complexes form per unit time
3. rate of reaction increases
4. it is the limiting factor
5. if sub conc is fixed then as the enzyme conc increases it will reach a point where it ceases to be the liming factor
6. this is bc all substrate molecule r occupied in their enzymes hence no more ES complexes can form and no products can form so the rate of reaction stays the same
7. substrate conc is now the limiting factor

Question 32

draw shape of graph of how enzyme conc affects enzyme acitivity

Answer 32

graph

Question 33

what is an inhibitor

Answer 33

substance that reduces or stops a reaction

Question 34

what is a competitive inhibitor

Answer 34

these are substances whole molecules have a similar shape to an enzyme’s substrate molecule and competes w the substrate molecule for the enzymes active site,
- it blocks the active site hence inhibits ES complexes and the reaction

Question 35

how does a competitive inhibitor work?

Answer 35

1. competitive inhibitor has similar shape to substrate
2. therefore is complementary to active site
3. binds to active site and competes for active sites against substrate molecules forming enzyme-inhibitor complexes
4. inhibitor are not changed in these complexes
5. presence of inhibitor prevents substrates from forming ES complexes and hence products so rate of reaction decreases as less active sites r available for substrates
6. more inhibitor molecules means more collide w active sites and hence inhibition is greater
7. can counteract this by increasing the sub conc

notes: usually competitive inhibitors r reversible
- competitive inhibitors that bind irreversibly r called inactivators

Question 36

draw the shape of a graph of the effect of a competitive inhibitor on the rate of reaction

Answer 36

graph

Question 37

what is a non-competitive inhibitor

Answer 37

non-competitive inhibitor molecules inhibit a reaction by NOT binding to the active site of a molecule but the ALLOSTERIC SITE of the enzyme
- this changes the shape of the active site of the molecule hence prevents ES complexes forming and hence inhibits the reaction

Question 38

how does a non-competitive inhibitor work?

Answer 38

1. non competitive inhibitor bind to the allosteric site of an enzyme
2. this changes the tertiary struc of an enzyme and hence the shape of the active site
3. this means that the active site is no longer complementary to the substrate molecule and hence ES complexes cannot form
4. this reduces the the max rate of reaction
5. however increasing substrate conc will not reduce the effect of non-competitive inhibitors to its uninhibited max
6. more inhibitor molecules r present the greater the inhibition (no ES complexes can form)
7. some inhibitors bind irreversibly and some bind reversibly

Question 39

draw shape in graph of how non-competitive inhibitors affect the rate of reaction

Answer 39

graph

Question 40

what bonds determine if the inhibitor will be reversible/irreversible

Answer 40

strong covalent bonds - irreversible

weak H bonds and ionic bonds - reversible

Question 41

how do metabolic pathways reactions regulate themselves

Answer 41

end-product inhibition

Question 42

how does end product inhibition work

Answer 42

1. metabolic pathway= series of connected metabolic reactions
2. product of the 1st reaction is the substrate for the 2nd reaction etc and this continues
3. until the product of the last enzyme is produced - when the conc of this is too high it attaches to the allosteric site of the first reactions enzyme
4. this changes the active sites shape and inhibits ES complexes forming hence inhibiting the reaction and hence the metabolic pathway (negative feedback)
5. this is non-competitive inhibition and is reversible
6. when conc of last product decreases it removes itself from the enzyme and hence the metabolic pathway can run again

Question 43

why is end-product inhibition important

Answer 43

• it stops the accumulation of too much end product and allows controlling of how much it gets made so energy is not wasted on unnecessary reactions

Question 44

give an example of end-product inhibition

Answer 44

• phosphofructokinase is an enzyme involved in metabolic pathway that breaks down glucose to make ATP
• ATP inhibits the action of phosphofructokinase so a high level of ATP inhibits that enzyme and hence the production of itself

Question 45

how do inactive precursors play a part in metabolic reactions

Answer 45

• some enzymes r synthesised as inactive precursors
• meaning they need some of their amino acids to b removed before their active site assumes the right shape and it can fully function
• e.g digestive enzymes r produced this way so they do not damage the body’s cells
• e.g trypsin is made in the small intestine in its inactive form trypsinogen
• once a small part of its molecule is removed it becomes active as trypsin
• e.g. pepsin is secreted as the inactive pepsinogen and this is converted into active pepsin by the action of hydrochloric acid in stomach

Question 46

give examples of metabolic poisons that act as enzyme inhibitors

Answer 46

1. cyanide

2. snake venom of the green mamba snake

Question 47

what is the difference between poison and venom

Answer 47

poison r toxins that r ingested and venom is introduced into the victims body by injection

Question 48

how does cyanide act as an enzyme inhibitor

Answer 48

• Potassium cyanide (KCN) inhibits aerobic respiration
• when ingested KCN is hydrolysed into a gas that can dissociate into H+ and CN- ions
• the CN- ions bind irreversibly to enzyme in mitochondira to stop the final stages of aerobic respiration

Question 49

how does snake venom act as an enzyme inhibitor

Answer 49

snake venom from the green mamba snake contains a chemical that inhibits the enzyme acetylcholinesterase (AChE)

• is important at neuromuscular synapses to break down neurotransmitter acetylcholine ACh
• if enzyme inhibited ACh stays attached to receptors on muscle membrane and hence muscles r kept contracted
• this causes paralysis - die of suffocation

Question 50

give examples of drugs that act as enzyme inhibitors

Answer 50

1. aspirin
2. ATPase inhibitors
3. ACE inhibitors
4. nucleoside reverse transcriptase inhibitors

Question 51

how does aspirin act as an enzyme inhibitor

Answer 51

• salicylic acid inhibits the enzymes that catalyse the formation of prostaglandins
• prostaglandins make nerve cells more sensitive to pain and increase swelling during inflammation
• aspirin reduces this pain and can also reduce blood clots

Question 52

how does ATPase inhibitors act as an enzyme inhibitor

Answer 52

• cardiac glycosides e.g. digitoxin inhibit sodium potassium pumps in the cell membranes of heart muscle cells
• this allows more calcium to enter the cells and hence increases muscle contraction
• they help treat heart failure and atrial arrhythmia
• extracted from purple foxglove leaves

Question 53

how does ACE inhibitors act as an enzyme inhibitor

Answer 53

these r drugs that inhibit ACEs which r enzymes that operates to increase blood pressure
hence the drugs lowers blood pressure in patients w hypertension/treats heart failure

Question 54

how does nucleoside reverse transcriptase inhibitors act as an enzyme inhibitor

Answer 54

• antiviral drugs such as zidovudine treat HIV patients and r nucleoside reverse transcriptase inhibitors
• they inhibit enzymes that make DNA using the viral RNA as a template

Question 55

describe how u would conduct an experiment that measures how fast the product of a reaction appears and use this to compare the rate of reaction under diff conditions

Answer 55

e.g how temp affects rate of reaction
(using catalase as example)
independent v = temp
dependent v = vol of O2 produced
1. set up boiling tubes containing same volume and conc of hydrogen peroxide - add buffer solution
2. set up rest of apparatus (bung connected to delivery tube connected to upside down cylinder in the water bath)
3. put each boiling tube in a water bath set to a diff temp e,g. 10/20/30 degrees along w another tube catalase
4. use a pipette to add same vol and conc of catalase in to each boiling tube then quickly attach bung and delivery tube
5. record how much oxygen is produced in first min of reaction using stopwatch to record time
6. repeat 3 times to work out mean vol of O2 produced
7. calculate mean rate of reaction at each temp - by dividing vol of O2 by time taken e.g 60 seconds units will b cm3/s

Question 56

describe how u would conduct an experiment that measures how fast the substrate is broken down and use this to compare the rate of reaction under diff conditions

Answer 56

e.g. how conc of substrate affect rate of reaction
( using amylase)
independent v = amylase conc
dependent = time it takes for starch to disappear
1. put drop of iodine into potassium iodide into each spotting tile
2. mix together known conc and vol of amylase and starch into test tube
3. using dropping pipette put drop of mixture into one of wells containing iodine solution at regular intervals (10secs)
4. observe resulting colour - should go from dark blue-black to orange brown if starch is present
5. u can see how fast amylase is working by recording how long it takes for iodine solution to no longer change colour when mixture of enzyme is added
6. repeat using diff concs of amylase
7. repeat 3 times for each conc

Question 57

how do you conduct an experiment to measure how pH affects rate of reaction

Answer 57

• add buffer solution to each pH to test tube

Question 58

how do you conduct an experiment to measure how substrate conc affects rate of reaction

Answer 58

• use serial dilutions to make substrate solutions w diff concs

Question 59

how do u estimate the initial rate of reaction

Answer 59

1. draw tangent at t=0
2. calculate gradient of tangent (change in y over change in x)
3. work out the units
   e. g. units of y /units of x

include diagram