Enzyme may be

used againEnzyme-

substrate

complex

E

S

P

E

E

P

Reaction coordinate

Enzymes

What are enzymes?

• Enzymes are globular proteins that

act as biological catalysts speeding up

chemical/metabolic reactions in

plants and animals they remaining

unchanged in the process

• They do this by lowering activation

energy

explain that enzymes are globular proteins that catalyse metabolic reactions; state that enzymes function inside cells (intracellular enzymes) and outside cells (extracellular enzymes)

Types of enzymes?• Enzymes can act within the cell

(intracellular enzymes) and they canact outside the cell (extracellularenzymes)

• Intracellular enzyme: ATP Synthase,RUBP carboxylase, DNA polymerase,etc

• Digestive enzymes: extracellularenzymes, lysozyme (an enzymeproduced by tear glands of the eyes),

explain that enzymes are globular proteins that catalyse metabolic reactions;

state that enzymes function inside cells (intracellular enzymes) and outside cells (extracellular enzymes)

Structure of enzymes • Enzymes are globular proteins that usually at the

tertiary or quaternary structure level.

• They possess an active site which is the point wheresubstrate molecules bind to before the enzyme-catalysed reaction can occur.

• This active site is specific for just one particularsubstrate, and complimentary to the shape of thesubstrate.

• The structure and shape of enzymes and their activesite is maintained by the interaction of differentamino acids within the proteins to form hydrogenbonds, ionic bonds, disulphide bonds andhydrophobic interactions.

explain that enzymes are globular proteins that catalyse metabolic reactions; state that enzymes function inside cells (intracellular enzymes) and outside cells (extracellular enzymes)

Mode of action of enzymes • Enzymes catalyse metabolic reactions by first binding to the substrate

at their active site to form an enzyme-substrate complex.

• The formation of this ES complex lowers the activation energy for the substrate(s) to react and to form product.

• Once the product(s) is(are) formed, they are released from the active site, and the enzyme is available to bind to another substrate(s)

explain the mode of action of enzymes in terms of an active site, enzyme-substrate complex, lowering of activation energy and enzyme specificity (the lock and key hypothesis and the induced fit hypothesis should be included);

The lock-and-key model • The enzyme has a specific shape into which the

substrate fits • The part of the enzyme where the substrate

attaches to is the active site. • The enzyme fits into the substrate the way a key

fits into a lock• The key is analogous to the enzyme and the

substrate analogous to the lock.• An enzyme-substrate complex is formed• Reaction occurs and the product (or products) is

formed• Product(s) differ in shape from the substrate and

so are released from the active site. • The site becomes free to bind to another substrate

explain the mode of action of enzymes in terms of an active site, enzyme-substrate complex, lowering of activation energy and

enzyme specificity (the lock and key hypothesis and the induced fit hypothesis should be included);

Enzyme may be used

againEnzyme-

substrate

complex

E

S

P

E

E

P

Reaction coordinate

The induced-fit model

• The enzyme and sometimes the substrate can change shape slightlyas the substrate molecule binds with the enzyme in order to ensure aperfect fit

• When the substrate combines with the enzyme, the enzyme changesits conformation, and develops the precise conformation that fits withthe substrate

• Activation energy is lowered and chemical reaction occurswithin/between the substrate(s)

explain the mode of action of enzymes in terms of an active site, enzyme-substrate complex, lowering of activation energy and enzyme specificity (the lock and key hypothesis and the induced fit hypothesis should be included);

Induced fit model• The induced-fit model is the

reason some enzymes can have two or more substrate since

those substrates have similar structure. E.g. hexokinase can have glucose or fructose as its

substrateHexokinase (a) without (b) with glucose substratehttp://www.biochem.arizona.edu/classes/bioc462/462a/NOTES/ENZYMES/enzyme_mechanism.html

Course of an enzyme-catalysed reaction

• Two ways of measuring the rate of an enzyme-catalysed reaction• Measure the rate of formation of products • Measure the rate of disappearance of substrates

• Reaction 1: Catalase H2O + O2

• Phase 1: very fast, large amount of products formed, due to large amount of substrate reacting

• Phase 2: slower; because the quantity of substrate is reducing

• Phase 3: reaction gradually stops, and no further products are formed; because no more substrates are available to bind to active site; substrates have been exhausted

• The rate of an enzyme catalysed reaction is always fastest at the beginning

investigate the progress of an enzyme-catalysed reaction by measuring rates of formation of products (for example, using catalase) or rates of disappearance of substrate (for example, using amylase)

Phase 1

Phase 2

Phase 3

Factors affecting enzyme catalysed reaction • For each factor we will consider the following:

• What happens when the factor is increased and why?

• What happens when the factor is reduced and why?

• Are the changes indefinite?

• How can the changes be reversed?

• How can the effects be represented on a line graph?

investigate and explain the effects of the following factors onthe rate of enzyme-catalysed reactions:

• temperature• pH (using buffer solutions)

• enzyme concentration• substrate concentration• inhibitor concentration

Enzyme Concentration• Increase in enzyme concentration results

in an increase in the rate of reaction

• This is because as enzyme concentrationincreases, more active sites are availableto bind with the substrate, more enzyme-substrate complex will be formed andmore products will be formed

• As long as there are plenty substrateavailable, increasing enzymeconcentration will result in increased rateof reaction.

investigate and explain the effects of the following factors onthe rate of enzyme-catalysed reactions:

•enzyme concentration

0 10 20 30 40 50

Enzyme Concentration

Initial

Rate of Reaction

• Increase in substrate concentrationresults in an increase in the rate ofreaction

• This is because as substrate concentrationincreases, more substrate molecules areavailable to bind to the active site, moreenzyme-substrate complex will be formedand more products will be formed.

• Increase in reaction rate due to increasedsubstrate concentration continues untilactive sites become saturated. At thatpoint, further increase in substrateconcentration will not result in a furtherincrease in the rate of reaction. Thereaction remains constant. The rate ofreaction at this point is known as Vmax

Substrate Concentration

investigate and explain the effects of the following factors onthe rate of enzyme-catalysed reactions:

•substrate concentration

Reaction

velocity

Substrate

concentration

Vmax

Enzyme affinity for substrates

• Different enzymes react with their respective substrates at different rates.

• The greater the affinity of an enzyme for its substrate, the faster the substratewill bind to its active site, and thus, the faster the rate of that enzyme catalysedreaction, compared to its reaction with another substrate for which it hasrelatively lower affinity for its substrate.

• If an enzyme A has a higher affinity for molecule A1 compared to molecule A2,enzyme A will react at a faster rate with A1 even if substrate concentration of A1is not high, compared to substrate A2. This is because of its high affinity

• With high affinity, a substrate does not need to have a very high concentration,for the rate of reaction to be high.

• This affinity of enzymes for substrate is related to a factor known as Michaelis-Menten constant (Km)

explain that the maximum rate of reaction (Vmax) is used to derive the Michaelis-Mentenconstant (Km) which is used to compare the affinity of different enzymes for their substrates

Michaelis Menten constant (Km)

• The concentration of substrate that isrequired for an enzyme to work at half of itsmaximum rate is known as Michaelis-Mentenconstant (Km)

• Or… Michaelis-Menten constant is thesubstrate concentration at which half of theenymes in a reaction system are saturated.

• The lower the value of Michaelis-Mentenconstant (Km), the greater the affinity of thatenzyme for its substrate.

• Some enzymes that catalyse reactionbetween two or more substrates havedifferent Km values for each substrates.

• The Km is determined using the Vmax and thisKm is also used to compare the affinity ofdifferent enzymes for their substrates

explain that the maximum rate of reaction (Vmax) is used to derive the Michaelis-Menten constant (Km) which is used to compare the affinity of different enzymes for their substrates

Enzyme Substrate Km (Mm)

Catalase H2O2 25

Hexokinase ATP 0.4

D-Glucose 0.05

D-Fructose 1.5

Chymotrypsine Glycytryosinlglycine 108

N-Benzoyltyrosinamide 2.5

Carbonic anhydrase HCO3 9

Significance of Km

• By knowing Km, one can determine whether a particular enzyme-substratesystem needs more enzymes or substrates to speed up the reaction

• An enzyme’s preference for different substrates can be comparedquantitatively. If an enzyme can catalyse a reaction with two similarsubstrates (e.g. hexokinase acting on glucose or fructose), it will prefer thesubstrate with lower Km value

• Activity of the same enzyme obtained from different organisms can also becompared

• Km values give an idea of the approximate concentration of substrate in aparticular system. High Km values indicate that there are higherconcentration of substrates in the system.

Temperature• As temperature increases, the rate of

enzyme catalyzed reaction increases.

• This increase occurs because as temperature increases, the kinetic energy of the enzymes and substrate molecules increases, they vibrate faster, they collide with each other at a faster rate, and the rate of formation of enzyme/substrate complex increases. Leading to reaction occurring at a faster rate.

• This increase continues until the optimum temperature of the enzyme is achieved.

• If temperature is increased further beyond optimum, the rate of reaction begins to reduce, until the reaction completely stops.

• The rate reduces because high temperature (higher than optimum) causes the denaturation of the enzyme’s tertiary structure, the active sites become distorted, substrate molecules can no longer bind to the enzyme, and reaction does not occur.

investigate and explain the effects of the following factors onthe rate of enzyme-catalysed reactions:

•temperature

Temperature / °C

Initial

Rate of Reaction

0 10 20 30 40 50

Q10 Denaturation

Effect of pH• Enzymes have a narrow range of

pH operation.

• Large changes in pH will cause denaturation of the enzyme, this changes/distorts the shape of the active site and substrate molecules cannot fit into the site.

• Enzyme/substrate complex cannot be formed, and thus, reaction will not take place.

• Changes in pH affects hydrogen bonds and ionic bonds that maintain the shape of the enzyme and active site.

investigate and explain the effects of the following factors on the rate of enzyme-catalysed reactions:•pH

Initial

Rate of

ReactionTrypsin

Pepsin

pH

1 3 5 7 9 11

Inhibition of enzyme-catalysed reactions• Inhibitors are chemical substances that slow down

the rate of enzyme activity.

• They do this by either binding to the active site of the enzyme and preventing the substrate from binding, or they bind to the allosteric site of the enzyme (and by that, alter the shape of the active site so that substrate cannot bind to the substrate)

• Inhibitors that bind to active sites are called competitive inhibitors. Inhibitors that bind to allosteric sites are called non-competitive inhibitors.

• Competitive inhibitors usually have a shape similar to the shape of the substrate, and that’s why they can fit into the active sites. However, non-competitive inhibitors have a different shape.

• Certain molecules that are final products of a metabolic pathway can act as inhibitors of the first enzyme in that pathway. This kind of inhibition is called end product inhibition

investigate and explain the effects of the following factors on the rate of enzyme-catalysed reactions:

•inhibitor concentration

Effects of inhibitors…

• Explanation 1: • If the concentration of inhibitors (both

competitive and non-competitive) is increased, the rate of enzyme catalyzed reaction reduces.

• This is because more competitors in the reaction medium will reduce the quantity of active sites available to bind to the substrate. Leading to reduced rate of reaction.

explain the effects of reversible inhibitors, both competitive and non-competitive, on the rate of enzyme activity

• Explanation 2: • If an enzyme catalyzed reaction is initiated and a fixed

volume/amount of competitive inhibitors is added, the rate of reaction is slowed down initially. But as substrate concentration is increased, the rate begins to increase again. And it will keep on increasing until the maximum rate is attained. However, the Michaelis-Menten constant will be reduced

• This is because increasing substrate concentration will make more substrates available to compete for the active site and thus they will be able to bind with the enzyme and react.

• Explanation 3: • If an enzyme catalyzed reaction is initiated, and a

fixed volume of non-competitive inhibitor is added, the rate of reaction is slowed down initially and even as substrate concentration increases, the maximum rate is not attained. However the Michaelis-Mentenconstant will not be changed.

Enzyme immobilisation• Enzyme immobilization is a process by which an

enzyme is attached to an inert, insoluble materialsuch as calcium alginate.

• This can provide increased resistance to changesin conditions such as pH or temperature.

• It also allows enzymes to be held in placethroughout the reaction, following which they areeasily separated from the products and may beused again - a far more efficient process and so iswidely used in industry for enzyme catalysedreactions

• Enzymes can be immobilised in the following steps: • amylase enzyme is mixed with a solution of sodium alginate

• this mixture is dripped (usually through a syringe) into a solution of calcium chloride

• the sodium ions are displaced by the calcium ions, resulting in the formation of hard, insoluble beads of calcium alginate, in which are trapped the molecules of amylase

• the alginate beads are left to harden further, following which they are rinsed

• After the beads are formed, they are normally placed in a suitablecontainer to create a column of beads.

• A suspension of starch can then be trickled down the column and collectedin a beaker. If the contents of the beaker are analysed, it is found to be asolution of maltase, without any starch being present i.e. the starch hasbeen hydrolysed by the amylase as it passed through the beads.

• By hydrolysing the starch in this way, there is no contamination of theproduct with enzyme – and the amylase remains in the beads, which canthen be used again.

• This is the case whenever it is possible to use immobilised enzymes. If theenzyme is not immobilised in this way, the product will contain enzymemolecules and a further stage will be required to recover them

Applications of enzyme immobilization

Advantages of immobilisation

• Enzyme can be recovered after use using a very coarse filter rather than a molecular filter

• Enzyme does not contaminate product

• Immobilisation may enhance stability (thermostability or pH-stability) of the enzyme molecule as it is supported

• Substrate can be easily passed through the enzyme several times

Measurement of glucose from urine samples

• Quantitative measurement of glucose needs to be done

in individuals with diabetes in order to determine the

quantity of insulin that they will need.

• This is done by testing a sample of urine with dipstick

• The dipstick has the enzyme glucose oxidase on its

surface – this oxidises the glucose into hydrogen

peroxide and gluconic acid.

explain the principles of operation of dip sticks containing glucose oxidase and peroxidase enzymes, and biosensors that can be used for quantitative measurement of glucose

Questions

1) The rate of an enzyme catalysed reaction is always fastest at the beginning; why? (4 marks)

Confused?Here are some links to fun resources that further explain enzymes:

• Enzymes Main Page on the Virtual Cell Biology Classroom of Science Prof Online.

• “How Enzymes Work” an animation from McGraw-Hill.

• Interactive Enzyme Tutorial and quiz from Life: The Science of Biology.

• “The Role Enzymes Play in the Body” from Enzymedica. These guys are selling supplements. I don’t endorse the supplement, but their video is very instructive regarding enzymes and digestion.

• “Assignment Discovery: Enzyme Catalysts” from HowStuffWorks, a Discovery Company.

• “Enzymes and Digestion” from About.com.

• “Bio Rad GTCA Song” musical advertisement for SsoFast™.

• And why not sing “Goodtimes with Enzymes” to the Chic song “Goodtimes.” See next slide for lyrics.

(You must be in PPT slideshow view to click on links.)

From the Virtual Cell Biology Classroom on ScienceProfOnline.com