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Biology > 3 Enzymes > Flashcards

3 Enzymes Flashcards

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1
Q

Enzymes

A
  • globular proteins that catalyse metabolic reactions inside cells (intracellular) or are secreted to catalyse reactions outside cells (extracellular) (and are not changed or used up)
  • enzymes do not make reactions occur, they speed them up
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2
Q

Intracellular enzymes

A

enzymes that remain IN the cell to catalyse metabolic reactions.

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3
Q

Extracellular enzymes

A

enzymes that are secreted and act OUT of the cell

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4
Q

Where are all enzymes synthesised?

A

Within the cell.

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5
Q

What type of proteins are enzymes and how does this affect their shape?

A

Globular proteins – meaning that they have a spherical shape.
Their shape is also therefore determined by their primary structure (sequence of amino acids).

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6
Q

Active site

A
  • the small region of an enzyme that is functional.
  • Only a few amino acids make up active site. - Active site forms a hollow depression within the much larger enzyme.
    -Area of active site directly involved with reaction known as catalytic site
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7
Q

How is a substrate molecule held in the active site?

A
  • Substrate molecule held by hydrogen bonds that temporarily form between R-groups of amino acids of active site and R-groups of substrate molecule.
    -Forms enzyme-substrate complex.
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8
Q

How would changes to amino acids in active site or tertiary structure disrupt catalysis?

A
  • These animo acids maintain the structure of the enzyme.
  • Changes could lead to the loss of the specific 3D shape of the active site.
  • Site would no longer be complementary to substrate, meaning catalysis cannot occur.
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9
Q

What are the two theories regarding how enzymes work?

A

The lock and key hypothesis.
The induced-fit hypothesis.

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10
Q

Why are enzymes soluble?

A
  • Amino acids with hydrophilic R-groups facing outwards to enzyme.
  • Makes enzyme soluble in e.g., blood or tissue fluid.
    -Demonstrates how enzyme structure is suited to function.
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11
Q

Explain the lock and key hypothesis?

A
  • Established concept of enzyme specificity.
  • Enzyme is lock, subtract is key.
    -Fit together perfectly without need for change.
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12
Q

Explain the induced fit hypothesis?

A
  • Newer and accepted theory
  • Enzyme active site is flexible and can undergo conformational change to change its form slightly to fit shape of substrate.
  • Because of this slight change, shape of active site and enzyme become FULLY complementary.
  • After reaction had been catalysed enzyme active site resumes original shape
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13
Q

Activation energy

A
  • Reactions need an initial ‘boost’ of energy to get them started. This is known as activation energy.
  • Means that an energy ‘hill’ must be overcome before rxn can proceed.
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14
Q

How do enzymes lower activation energy?

A
  • Enzymes holding substrates together for easier bond formation
  • Exposing bonds (Some molecules require bonds to first be broken before new ones can be formed to form product. Enzymes expose these bonds, making it easier for them to break).
  • Holding substrate in a way that strains bonds so they can be broken
  • Providing hydrophobic region for reaction involving non-polar substances. Enzymes can provide a special environment, called a hydrophobic region, where these reactions can occur more easily.)
  • Transfer of electrons (Enzymes can help facilitate this transfer by providing a pathway or a “bridge” for the electrons to move along.)
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15
Q

What is enzyme specificity?

A
  • Idea that enzyme is specifically complementary to a substrate molecule, allowing the enzyme-substrate complex to form and the reaction to be catalysed.
    -The induced fit hypothesis resulted from wondering why one enzyme could work on two or similar substances.
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16
Q

What are the two requirements for an enzyme to ‘work’ and how are enzyme reactions influenced?

A
  • Enzyme must come into physical contact with substrate.
  • Enzyme must have active site which fits substrate.
  • Enzyme-controlled reactions/rate of catalysis is influenced by manipulating one of these two factors.
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17
Q

How to measure enzyme-catalysed reactions?

A
  • Enzyme catalysed rxns are measured through measuring time-course.

Either measure:
- Time course of formation of products of rxn e.g., volume of oxygen produced when catalase acts on hydrogen peroxide.
- Time course of disappearance of substrate of rxn
e.g., the reduction in concentration of starch when acted upon by amylase.

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18
Q

Enzyme assay

A

an experiment used to determine the activity of an enzyme

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19
Q

Explanation of enzyme action graph curve?

A
  • At first substrate is abundant. No product.
  • Easy for substrate molecules to come into contact with empty active sites.
  • Quantity of substrate decreases – being broken down.
  • Quantity of product increases.
  • Product being produced per unit time decreases because there are fewer substrate molecules (they are mostly broken down into product). Not all active sites able to be filled.
  • Rate of rxn continues to slow as substrate concentration decreases.
  • Graph flattens out. All substrate used up. No new product able to be produced. 0-
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20
Q

When will an enzyme perform at its maximum rate (Vmax) during a rxn?

A

At the beginning when there is an abundance of substrate molecules to fill active sites.

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21
Q

How to measure rates of formation of products using catalase?

A
  • Hydrogen peroxide (2H202), substrate, catalase solution and enzyme are mixed together.
  • Volume of oxygen produced recorded at set time intervals.
  • Can use gas syringe to measure volume of oxygen produced OR could use method of downward displacement of water.
  • Independent variable is time. Plot on x-axis. Should be at least 5 time readings.
  • Dependent variable is volume of oxygen produced. Plot on y axis.
  • Standardised/controlled variables include temperature and concentration of solutions.
  • A control run should be performed to validify the experimental results.
    E.g., use the same quantity of boiled (denatured) catalase enzyme to test that it really is catalase causing oxygen to be produced/catalysing the reaction.
  • Other variables – like how hydrogen peroxide when added will displace some air in the conical flask and collect in the gas syringe – must be taken into account.
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22
Q

How to measure rates of disappearance of substrate using amylase?

A
  • Create KI solution.
  • Changes in colour of solution (with addition of enzyme and substrate) over time reflects decrease in substrate (due to hydrolysis facilitated by enzyme).
  • End point is when solution has changed completely from blue-black to orange.
  • Use spotting tile and with dropper add one drop of KI solution to each well.
  • At time 0 add one drop of enzyme-substrate solution to first well.
  • Record colour.
  • Repeat sampling in intervals (comparative data).
  • Record time when end point was reached.
  • Rate of reaction can be calculated non-numerically through: 1/t (time taken for reaction to finish).
  • Have a control. Variables should be standardised. Temp and concentration.
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23
Q

What must you do since iodine does not dissolve in water

A

you must add it to solution. Iodine + potassium = potassium iodide. (KI solution)

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24
Q

Colorimeter

A

light-sensitive piece of equipment that gives quantitative measurement of coloured solution

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25
Q

How does a colorimeter work?

A
  • Solution placed into cuvette (small rectangular cylinder). Cuvette is CLEAR on two opposite sides and FROSTED on the other two.
  • Cuvette placed into sample well of colorimeter. Beam of light aimed through CLEAR sides of cuvette. Some light is ABSORBED by solution – other light is TRANSMITTED.
  • Light reaches a sensor. Numerical reading is obtained.
  • Transmission value given in percentage transmission or an absorbance value is given in absorbance units (au).

Dark colour = higher absorbance, lower transmission.
Lighter colour = higher transmission, lower absorbance.
- Different wavelengths/colours can be used but the best choice is the wavelength most absorbed by a colour.
- Colorimeter is calibrated/standardised using blank solution in cuvette – e.g., distilled water.

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26
Q

How to control temperatures of solutions

A

by using a digitally controlled water bath is a great idea.

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27
Q

When measuring hydrolysis of starch with iodine what will happen to absorbance?

A

absorbance will decrease as blue-black colour changes to orange.

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28
Q

How to determine concentration of starch in a sample cuvette?

A
  • Prepare a calibration curve by using a colorimeter to obtain readings for known concentrations of starch solution.
  • Plot absorbance (y) against concentration of starch (x) and draw in calibration curve. - Will allow absorbance readings to be ‘directly converted’ to starch concentrations.
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29
Q

Factors that affect enzyme action

A
  • Temperature and pH
  • Enzyme and substrate concentration
  • Effects of inhibitors on enzyme action
  • Immobilised enzymes
30
Q

How does temperature affect enzyme rate of reaction?

A
  • Rise in temp increases kinetic energy of molecules
  • Random collisions between substrate and enzyme can occur more frequently
  • Rate of rxn increases
  • Shown on graph by rising curve.
31
Q

How does enzyme denaturation occur if the temp is increased too much?

A
  • Rise in temp increases energy of atoms that make up enzyme
  • Atoms begin to vibrate – this causes bonds (especially weak bonds like hydrogen bonds) to break
  • GRADUALLY the shape of active site begins to change. First substrate fits less easily, slows rate of rxn down (45 degrees C).
  • Tertiary structure changes so much that active site is not complementary to substrate. Enzyme is said to be denatured. (60 degrees C).
  • Shown on graph by falling curve.
32
Q

When do humans enzymes start denaturing and are denatured?

A

human enzymes START denaturing at about 45 degrees C, denatured at 60 degrees C

33
Q

Why would a human body temp above 37 degrees C be unsuitable for enzyme function?

A
  • Too much food would be required to maintain a higher temperature.
  • Other proteins could be denatured at a higher temperature.
  • Any rises in temp during e.g., illness could denature enzymes.
34
Q

How does too little heat affect enzyme activity?

A

Enzymes become inactive at lower temperatures – not denatured and can be reversed with increase of temp.

35
Q

What is pH referring to?

A

Hydrogen ion concentration. pH = potential hydrogen.

36
Q

How do small changes in pH affect enzyme activity?

A
  • Small changes in pH can influence the R-group of an animo acid, making it more or less likely to donate or accept a hydrogen ion.
  • The R-group can then become ionised and disrupt the shape and charge distribution of the active site, influencing how well the enzyme can bind to its substrate.
  • Small changes in pH affects efficiency of catalysis.
  • Ability to accept/donate hydrogen ions makes amino acid R-groups ionisible. (Ionisible R-groups).
  • If amino acid R-group loses or gains a hydrogen ion we can say it has become ionised.
  • Therefore, pH can change ionisation state of R-group.
37
Q

How do large changes in pH affect enzyme activity?

A
  • Cause hydrogen and ionic bonds to break.
  • Changes tertiary structure of enzyme, which changes its shape as a whole and leads to active site becoming deformed.
  • Large changes of pH causes enzyme denaturation.
38
Q

Buffer solutions

A

solutions that can be used to prevent fluctuations in pH (often by neutralising acid or base being added

39
Q

Steps to follow/determine when doing a chemical experiment (with enzymes)?

A
  • Choosing appropriate chemicals
  • Choosing correct apparatus
  • Identifying independent variable
  • Identifying dependent variable
  • How to change/manipulate independent variable
  • What range and spacing is to be used for independent variable; number of values at which dependent variable should be recorded
  • How frequently dependent variable should be measured
  • Whether or not experiment should be repeated
  • Which variables are possible to standardise
  • Whether or not a control can be performed
40
Q

How to check the pH of a solution?

A
  • Use a pH probe and meter
  • Use universal indicator paper
41
Q

When is an enzyme controlled rxn always most rapid?

A

At the start when there is enough substrate to as many active sites as possible.

42
Q

Why can enzymes work efficiently even at low concentrations?

A

They are not used up or changes in a rxn, so after catalysing substrate they RELEASE product and can act on a new substrate.

43
Q

What is outcome of increase in enzyme concentration (if there is an excess of substrate)?

A
  • As long as there is an EXCESS of substrate molecules, an increase in enzyme concentration will lead to a proportionate increase in the rate of rxn.
  • Shown by straight upwards line on graph.
44
Q

What is outcome of increase in enzyme concentration if no excess of substrate?

A
  • If there is an increase in enzyme concentration but no corresponding increase in substrate concentration then this will have no effect of rate of rxn.
  • Rate of rxn will stabilise at constant level and will be shown by graph levelling off.
  • Why? Because available substrate already being used as rapidly as it can by existing enzymes.
45
Q

Enzyme concentration or substrate concentration can act as a?

A

limiting factor of rate of rxn in enzyme-controlled reactions.

46
Q

the Vmax of a rxn

A
  • when all enzyme active sites are fully saturated with substrate molecules.
  • After Vmax has been reached addition of any more substrate will have no effect on reaction.
    -Reaction will level off (when substrate is in excess without increase in enzyme concentration).
47
Q

Vmax

A
  • refers to maximum rate/maximum velocity of reaction of an enzyme, when all active sites are saturated by substrate.
  • Used to derive Km (Michaelis-Menten constant) – which is used to compare the affinity of different enzymes for their substrates.
48
Q

Km (Michaelis-Menten constant)

A
  • substrate concentration needed for an enzyme reaction to proceed at HALF its Vmax.
  • Measures affinity of an enzyme for its substrate.
49
Q

How can the Michaelis-Menten constant be used to compare enzyme affinity?

A

E.g., Consider 2 enzymes: enzyme X and enzyme Y.

Enzyme X has a higher Km than enzyme Y.

  • Means that enzyme X needs more substrate to reach half of its maximum rate of rxn, and to reach Vmax.
  • Means that enzyme Y has a higher enzyme affinity.
  • It can more easily bind to its substrate at lower concentrations so doesn’t need as much substrate.
50
Q

Enzyme affinity

A
  • How much an enzyme likes to bind with its substrate.
  • High enzyme affinity usually means the enzyme can grab onto its substrate easily, even at low concentrations of the substrate.
51
Q

Enzyme inhibitors

A

substances that directly or indirectly interfere with functioning of active site of enzyme, and therefore reduce enzyme activity

52
Q

What are the two types of reversible enzyme inhibitors?

A
  • Competitive
  • Non- competitive
53
Q

Competitive inhibitors

A

inhibitor binds to active site of enzyme instead of substrate

54
Q

Non-competitive inhibitors

A

inhibitor binds to enzyme at allosteric site, changes the shape of active site

55
Q

How do competitive inhibitors work?

A
  • Molecular shape similar to substrate. Mimics substrate and binds to active site.
  • Compete with substrate molecules for available active sites.

If concentration of substrate is increased, effect of inhibitor is reduced.
- Because bond to active site is temporary, after inhibitor leaves substrate molecule can take its place.
- Means that all substrate molecules will find an active site eventually
- will take long if high inhibitor concentration and low substrate concentration.
- This will lower rate of rxn.

56
Q

How do non-competitive inhibitors work?

A
  • Attach at allosteric site.
  • Inhibitor alters shape of enzymes active site so that its catalytic activity is reduced/non-functional. Enzyme cannot function.
  • In some cases substrate can still enter active site, but enzyme will still not catalyse rxn.
  • since inhibitor and substrate molecule are not competing for the same active site, an increase in substrate molecule will have NO effect.
57
Q

Allosteric site

A
  • a specific region on a protein molecule
    other than the active site, where small molecules or ions can bind to regulate the protein’s activity
58
Q

How can inhibitors be helpful?

A
  • Allosteric regulators regulate metabolic reactions within cell.
  • These are reversible inhibitors that bind to allosteric site.
  • Control activity of enzyme when enough product has been made.

They do this through end-product inhibition.
Substrate -> enzyme 1 -> product 1 -> enzyme 2 -> product 2 -> enzyme 3 -> product 3

  • In this example, when there is enough product 3 an allosteric regulator will inhibit enzyme 1.
  • Leads to less product 3 forming.
  • When more product 3 is required inhibition is removed and pathway continues.
59
Q

How can inhibitors be harmful?

A

Permanent allosteric inhibitors can damage cell health and may lead to cell death

60
Q

How to investigate the effect of enzyme inhibitors?

A
  • Rate of reaction must be determined over a range of substrates – each with and without inhibitor.
  • Main variables to be standardised: temperature, enzyme concentration, volume of enzyme solution, volume of substrate solution, pH, concentration of inhibitor.
61
Q

How does competitive inhibition affect Vmax and Km?

A
  • When the substrate concentration is higher, effect of comp inhibitor decreases.
  • Vmax is reached.
  • Km increases, as more substrate is needed for reaction to reach half its Vmax because inhibitor is competing with substrate.
  • Enzyme has a lower affinity for substrate.
62
Q

How does non-competitive inhibition affect Vmax and Km?

A
  • Enzyme has same affinity for substrate because inhibitor is not competiting for active site.
  • Inhibitor and substrate can bind at same time.
  • Vmax is not reached.
  • Km remains the same because inhibitor does not interfere with substrates ability to BIND to enzyme.
63
Q

Why is Vmax still reached for competitive inhibition

A
  • For competitive inhibitors which form an enzyme-inhibitor complex, inhibition can still be overcome by increasing the substrate concentration.
  • At high enough concentrations enough substrate molecules will bind to the active site and surpass the effect if the inhibitor.
  • Therefore, Vmax can still be reached because the inhibitor doesn’t directly interfere with or affect the enzyme’s catalytic activity or ability.
64
Q

Why is Vmax not reached for non-competitive inhibition?

A
  • non-competitive inhibitors, in binding to the allosteric site, cause a conformational change that reduces catalytic activity, directly interfering with an enzyme’s ability to catalyse a reaction.
  • Increasing the substrate concentration will not reduce the inhibitor effect as the substrate can bind to the enzyme whether or not the inhibitor is there.
  • Even at high concentrations of substrate the inhibitor still binds to the enzyme and reduces its catalytic activity, meaning Vmax can’t be reached.
65
Q

What is the purpose of immobilising enzymes?

A

so that they can be retained and reused instead of discarded after catalysing a reaction.

66
Q

How can enzymes be immobilised (trapped) using sodium alginate beads?

A
  • Mix enzymes that need to be immobilised with a solution of sodium alginate.
  • Tiny droplets of this mixture are added to solution of calcium chloride. Use a syringe and add drops one at a time.
  • Reaction occurs between sodium alginate-enzyme mixture and calcium. Causes calcium ions to replace the sodium ions.
  • As a result, jelly-like beads are formed with enzyme trapped inside.
67
Q

How can immobilised enzymes catalyse reactions?

A
  • Beads are packed into a long column in a vessel.
  • Substrate is poured in at top.
  • As substrate trickles down column, it is converted to product.
68
Q

Why can immobilised enzyme reactions not continue indefinitely?

A

Impurities will accumulate.

69
Q

What are the advantages of using immobilised enzymes or cells?

A
  • Enzyme can be used repeatedly – cheaper.
  • Calcium alginate beads trapping enzymes act as a buffer against changes in pH and temperature. Makes ‘shelf-life’ of enzyme longer.
  • Purer product is formed because trapped enzymes can’t contaminate it.
  • In whole cell immobilisation, number of enzymes can at together at same time in a single process – very effective.
  • Protection given by entrapment means that reaction can occur at a higher temperature, increasing rate of reaction, increasing productivity.
  • Process can be carried out on continual basis – no need to continuously stop and remove enzyme from product.
70
Q

Free enzyme and immobilised same enzyme will likely have

A

different optimum temp and pH

71
Q

Example of immobilised enzyme usage

A
  • to remove lactose from milk.
  • Immobilised enzyme B-galactosidase.
  • Catalyses hydrolysis of lactose to glucose and galactose.
  • Prescence of glucose specifically tested for by glucose dipstick.

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