Enzymes

Class 11 Biology, unit 3, book pages 83–100

Practise 76 MCQs Take the unit quiz Review 55 cards

Enzymes are biological catalysts, mostly globular proteins, that speed up the many chemical reactions of metabolism without being used up. This unit explains the active site and cofactors, how enzymes work (lock and key and induced fit, lowering the energy of activation), what affects their rate (temperature, pH, enzyme concentration), how they are inhibited or regulated, and how they are classified. It is the base for respiration, photosynthesis and digestion, and is examined through definitions, differences, graphs and labelled diagrams.

Learning outcomes (14)

Identify the role and component parts of the active site of an enzyme.
Differentiate among the three types of co-factors i.e., organic, ions, prosthetic group and co-enzymes with examples.
Explain the mechanism of enzyme action with the help of the induced fit model, including comparing with lock and key model.
Explain enzyme catalysis with examples of specific reactions.
Define energy of activation and discuss through graph how an enzyme speeds up a reaction by lowering the energy of activation.
Explain the effect of temperature on the rate of enzyme action with example of human and thermophilic bacteria.
Investigate the effect of pH on enzyme activity. Compare the optimum pH of different enzymes like trypsin, pepsin, and papain.
Demonstrate that the concentration of enzyme affects the rate of enzyme action.
Describe enzyme inhibition, its types and its significance with examples.
Name the molecules which act as inhibitors.
Categorize inhibitors into competitive and non-competitive inhibitors.
Explain feedback inhibition.
Classify enzymes on the basis of reactions catalyzed (oxido-reductases, transferases, isomerases and ligases).
Classify enzymes on the basis of substrates they use (lipases, diastase, amylase, proteases etc.).

3Enzymes and metabolism

There is a complete check and balance on the chemistry of a cell. It shows itself in the many enzymatic reactions going on inside the cell. The sum of all the chemical reactions going on in a cell is called metabolism. These reactions must be carried out very quickly so that their products can be used in the various life activities of the cell. Enzymes are what make them fast.

Metabolism: The sum of all the chemical reactions going on in a cell.
Enzymes: Biological catalysts. They speed up biochemical reactions without being consumed. Enzymes exist in the cell as colloids.
Enzyme (definition): Biological polymers that catalyse biochemical reactions.

3.1Enzyme structureOften examined

All enzymes are globular proteins made up of one or more polypeptides. The only exceptions are the ribozymes, which consist of RNA and are found in ribosomes. For example, peptidyl transferase is a ribozyme which forms the peptide bond during protein synthesis.

An enzyme consists of a linear chain of amino acids. The chain folds to form the three-dimensional structure of the enzyme, and this shape is what makes the active site possible.

Own diagram: a linear chain of amino acids folds into the compact, three-dimensional (globular) enzyme, which carries the active site.

Ribozyme: An enzyme that consists of RNA instead of protein. Peptidyl transferase, which forms the peptide bond during protein synthesis, is an example.

3.1.1Components of an active site of an enzymeOften examined

The catalytic activity of an enzyme is located in its active site. The active site is a specific charge-bearing, three-dimensional cavity. The substrate (the reactant that is to be converted into product) is attached to the active site by non-covalent interactions such as hydrogen bonding and hydrophobic interactions.

The active site consists of 3 to 12 amino acids. They may be scattered along the polypeptide, but secondary and tertiary folding of the protein brings them together in a particular fashion. For example, the active site of aldolase consists of glycine, histidine and alanine.

The shape of the active site is designed according to the substrate, so only a particular substrate can attach to it. Sometimes a related substrate can also bind.

Figure 3.1: Active site. (a) Which substrate fits the active site? (b) Grouping of amino acids of a polypeptide during the formation of the tertiary structure.

Binding site and catalytic site

An active site has two functional regions. Some of its amino acids make the binding site: they hold the substrate. The other amino acids make the catalytic site: they bring about the conversion of the substrate into product (catalysis).

Own diagram: the two regions of an active site. The binding site holds the substrate; the catalytic site converts it into product.

Binding site and catalytic site
Point	Binding site	Catalytic site
Job	Attaches the substrate to the active site	Converts the substrate into product (catalysis)
Made of	Some amino acids of the active site	The other amino acids of the active site
Result	Enzyme-substrate complex forms	Substrate is changed; product is formed and released

Cofactors, apoenzyme and holoenzyme

Some enzymes also need a non-protein part, the cofactor. The cofactor is not only responsible for attaching the substrate to the active site; it also takes part in the catalytic process. The final shape of the active site is established only after the cofactor is attached.

An enzyme that needs a cofactor becomes active only when the cofactor is combined with it. Such an active enzyme is called a holoenzyme. If the cofactor is not available, the remaining protein part is catalytically inactive and is called an apoenzyme.

Figure 3.2: Structure of an enzyme. Without the cofactor attached, the protein is not active. Cofactor binding activates the protein.

Apoenzyme and holoenzyme
Point	Apoenzyme	Holoenzyme
Composition	Protein part only	Protein part plus cofactor
Activity	Catalytically inactive	Catalytically active
Active site	Final shape not yet established	Final shape established after the cofactor is attached

Enzymes that do not need a cofactor can also exist in active and inactive states. Pepsin is an example. The gastric glands of the stomach wall secrete it in an inactive state, the pepsinogen. In this state an additional polypeptide fragment is attached to its active site, so substrate cannot bind and the enzyme stays inactive.

Activation of pepsinogen

Pepsinogen is secreted by the gastric glands as an inactive enzyme (the apoenzyme form). An extra polypeptide fragment blocks its active site.
Pepsinogen is exposed to HCl, as in the stomach cavity.
The additional polypeptide fragment is removed.
The inactive pepsinogen changes into its active form, the pepsin (the holoenzyme form), which can now bind its substrate.

Own diagram: pepsinogen is activated to pepsin when HCl removes the polypeptide fragment that blocks the active site.

3.1.2Types of cofactorsOften examined

There are three types of cofactors: inorganic ions, organic molecules and the prosthetic group. The organic cofactors are either coenzymes or prosthetic groups.

Own diagram: the types of cofactor. Inorganic ions and coenzymes are detachable; a prosthetic group is permanently attached.

Inorganic ions

The inorganic cofactors are different metallic ions such as $Fe X^{2 +}$ , $Mg X^{2 +}$ , $Cu X^{2 +}$ and $Zn X^{2 +}$ . They are attached to the enzyme only when the substrate gets bound, so they are detachable cofactors. Such cofactors are also called activators.

Glucose + ATP Hexokinase + Mg^{2 +} Glucose-6-phosphate + ADP

Magnesium ions as activator of hexokinase

Organic molecules

The organic cofactors are either coenzymes or prosthetic groups. The coenzymes are derivatives of vitamins. For example, ATP, $NAD X^{+}$ and FAD are common coenzymes (the book writes FAD $^{+}$ ). Like inorganic cofactors, they are attached to the enzyme when the substrate gets bound, so they are also detachable cofactors.

Prosthetic group

A prosthetic group is a covalently bonded part of an enzyme. It is permanently attached to the enzyme and does not detach after the completion of the reaction. An iron-containing porphyrin ring attached to some enzymes such as the cytochromes is an example of a prosthetic group.

Ethyl alcohol Alcohol dehydrogenase + NAD^{+} Acetaldehyde + NADH_{2}

NAD+ as coenzyme of alcohol dehydrogenase

Prosthetic group and coenzyme
Point	Prosthetic group	Coenzyme
Attachment	Covalently bonded; permanently attached	Attached when the substrate binds; detachable
After the reaction	Does not detach	Leaves the enzyme
Nature	Organic cofactor, for example the iron-containing porphyrin ring of cytochromes	Organic cofactor derived from a vitamin, for example NAD $^{+}$ , FAD, ATP

Inorganic and organic cofactors
Point	Inorganic cofactor	Organic cofactor
Nature	Metallic ions such as $Fe X^{2 +}$ , $Mg X^{2 +}$ , $Cu X^{2 +}$ , $Zn X^{2 +}$	Organic molecules: coenzymes or prosthetic groups
Other name	Activators	Coenzymes (detachable) or prosthetic group (permanent)
Attachment	Detachable; bound when the substrate gets bound	Coenzymes are detachable; a prosthetic group is permanent
Example	$Mg X^{2 +}$ with hexokinase	NAD $^{+}$ with alcohol dehydrogenase

3.2Mechanism of enzyme actionOften examined

The substrate first binds to the active site of the enzyme to form an enzyme-substrate (ES) complex. Then the substrate is converted into product while it is attached to the enzyme (the EP complex). Finally the product is released, which allows the enzyme to start all over again.

enzyme E + substrate S \to enzyme-substrate complex ES \to enzyme-product complex EP \to enzyme E + product P

Enzyme action in outline

The cycle of an enzyme

The enzyme (E) and its substrate (S) meet. The substrate attaches to the active site.
An enzyme-substrate (ES) complex is formed.
While attached, the substrate is converted into product: the enzyme-product (EP) complex.
The product (P) is released.
The enzyme is unchanged and is ready to catalyse a new reaction.

3.2.1Models of enzyme actionOften examined

The mechanism of enzyme action is explained with the help of two models: the lock and key model and the induced fit model.

Lock and key model

Emil Fischer proposed the lock and key model in 1894. According to it, the enzyme is a lock and the substrate is a key. A key has a shape similar to that of the keyhole of its lock; in the same way the substrate has a shape similar to the active site of the enzyme. The substrate binds tightly to the active site, just like a key into its lock. If the shape of the substrate and the active site are not similar, the substrate cannot bind. The enzyme is a rigid structure, and the shape of the active site does not change or modify during binding. (In some books and diagrams the key is the enzyme and the lock is the substrate.)

Figure 3.3: Lock (enzyme) and key (substrate). (a) Separate. (b) Lock-key complex.

Figure 3.4: Fischer's lock and key hypothesis of enzyme action. The active site is rigid and does not change in shape.

The notched portion of the key is equivalent to the active site on the enzyme. This reflects that enzymes are highly specific in their action and each enzyme can carry out only one particular reaction. Enzymes that work according to this model are called non-regulatory enzymes. However, only a very small number of enzymes follow this model, for example sucrase and maltase.

The ability of an enzyme to catalyse one specific reaction is perhaps its most significant property. Although many enzymes show a broad range of specificity towards the substrate they catalyse, when one enzyme can catalyse only one substrate and essentially no others it is called absolute specificity, for example urease.

Urea + Water Urease Ammonia + Carbon dioxide

Induced fit model

Koshland proposed the induced fit model in 1959. According to it the active site is flexible, so it is modified as the substrate interacts with the enzyme. The amino acids that make up the active site are moulded into a precise shape, which enables the enzyme to perform its catalytic function more effectively. The change induced in the shape of the active site is responsible for the conversion of substrate into product. When the reaction is completed, the active site regains its original shape.

This flexibility of the active site allows more than one type of related substrate to be attached to the active site, and therefore an enzyme can carry out more than one type of related reaction. An example is carbonic anhydrase, which can add $O X_{2}$ to haemoglobin as well as control the formation of carbonic acid and bicarbonates in blood. Enzymes that follow the induced fit mechanism are called regulatory or allosteric enzymes, for example hexokinase.

Hb + 4 O_{2} Carbonic anhydrase Hb (O_{2})_{4} (oxyhaemoglobin)

CO X_{2} + H X_{2} O Carbonic anhydrase H X_{2} CO X_{3} Carbonic anhydrase H X^{+} + HCO X_{3} X^{-}

Figure 3.5: Koshland's induced fit model of enzyme action. The active site is flexible and changes shape as the substrate binds.

Difference between induced fit and lock and key model

In the induced fit model the active site of the enzyme does not completely fit the substrate at first. In the lock and key model the active site of the enzyme is the complement of the substrate, so it precisely fits the substrate. In the induced fit model the active site has to undergo a conformational change to improve binding. The lock and key model describes the specificity of the active site of the enzyme for a particular substrate.

Lock and key model and induced fit model
Point	Lock and key model	Induced fit model
Proposed by	Emil Fischer (1894)	Koshland (1959)
Active site	Rigid; its shape does not change	Flexible; moulded when the substrate binds, then returns to its original shape
Fit with substrate	Complement of the substrate; fits precisely from the start	Does not completely fit at first; improves by a conformational change
Enzyme type	Non-regulatory (few enzymes), for example sucrase, maltase	Regulatory or allosteric, for example hexokinase
Specificity	One substrate, one reaction (absolute specificity), for example urease	More than one related substrate and reaction, for example carbonic anhydrase

Worked example: which model fits the enzyme?

Urease acts only on urea. Carbonic anhydrase acts on the reaction of carbon dioxide with water and also helps haemoglobin to take up oxygen. For each enzyme, name the model of enzyme action it follows, say whether it is a regulatory or a non-regulatory enzyme, and give the reason.

Enzyme	What it does	Model	Type	Reason
Urease	Urea + water gives ammonia + carbon dioxide; no other substrate	Lock and key	Non-regulatory	Absolute specificity: one enzyme, one substrate. A rigid active site fits only urea.
Carbonic anhydrase	$CO X_{2} + H X_{2} O H X_{2} CO X_{3}$ and $H X_{2} CO X_{3} H X^{+} + HCO X_{3} X^{-}$ (book), and oxygen uptake by haemoglobin	Induced fit	Regulatory (allosteric)	The active site is flexible and is moulded by each substrate, so one enzyme can carry out more than one type of related reaction.

Decide from the specificity. One substrate and one reaction only points to a rigid active site that is the complement of the substrate: the lock and key model.
More than one related reaction needs an active site that can change shape to suit each substrate: the induced fit model.
Link the model to the enzyme type. Lock and key enzymes are non-regulatory enzymes; induced fit enzymes are regulatory (allosteric) enzymes, for example hexokinase.

Urease: lock and key, non-regulatory. Carbonic anhydrase: induced fit, regulatory (allosteric).

3.2.2Energy of activationOften examined

Molecules do not react with one another unless they are activated in some way. The energy that must be added to cause molecules to react with one another is called the energy of activation. In a non-living system we use heat as the energy of activation, to increase the number of effective collisions between molecules.

In living systems a large amount of heat cannot be used as the energy of activation. Why? All living cells and organisms are mainly composed of temperature-sensitive protein molecules. About 1,000 chemical reactions are being carried out in a cell at any time, and the energy of activation required for such a large number of reactions cannot be provided by the living system.

The living system works in isothermal conditions (at one temperature). The excited state of molecules or reactants is achieved by a biochemical process. Enzyme (E) reacts with reactant (A) to form an AE transitional complex. The energy level of the AE complex reaches the energy level of reactant B. The AE complex then reacts with reactant B to form AB, and the enzyme (E) is released.

A + E \to AE complex + B \to AB + E

An enzyme decreases the energy of activation. The energy of activation is the energy required to break the existing bonds and begin the reaction; an enzyme greatly reduces the activation energy necessary to start a chemical reaction.

Figure 3.6: Energy of activation. Enzymes speed up chemical reactions because they lower the amount of energy required to activate the reactants. The enzyme lowers the activation energy but the reactants and products stay at the same energy levels.

How the graph shows an enzyme speeding up a reaction

Read the graph from left to right. The reactants start at one energy level.
The reactants must climb an energy hill, the energy of activation, before they can change into products. Without an enzyme the hill is high.
With the enzyme the hill is lower, so a larger number of reactant molecules have enough energy to react in the same time: the reaction is faster.
Both curves end at the same level of the products. The change in free energy between reactants and products is the same with and without the enzyme.

3.3Factors affecting the rate of enzymatic action

The rate of an enzymatic reaction is measured by the amount of substrate changed, or the amount of product formed, during a period of time. The external conditions that affect the rate of enzyme reactions are temperature, pH, concentration of enzyme and substrate concentration.

The four external conditions
Factor	How it affects the rate	Learn in
Temperature	Rate rises with temperature up to the optimum, then falls because the enzyme is denatured	Temperature
pH	Each enzyme works best at its optimum pH; changes alter the charges that hold its shape	pH
Enzyme concentration	With plenty of substrate, rate is directly proportional to enzyme concentration	Enzyme concentration
Substrate concentration	Rate rises with substrate, then levels off when the enzyme is saturated	Substrate concentration

3.3.1TemperatureOften examined

Heating increases molecular motion. The molecules of the substrate and enzyme move more quickly, so the probability of a reaction occurring is increased. An increase of just 10 °C in the existing temperature doubles the rate of reaction, but this effect remains only up to a certain limit.

The temperature that promotes maximum activity is called the optimum temperature. If the temperature is increased above this level, the rate of the reaction decreases despite the increasing frequency of collision. This is because the secondary and tertiary structures of the enzyme have been disrupted, so the enzyme unfolds and the precise structure of the active site is gradually lost. The enzyme is said to be denatured. The temperature that causes denaturation of the enzyme is called the maximum temperature. The bonds most sensitive to temperature change are the hydrogen bonds.

Temperature (^{\circ} C)

Rate

Optimum temperature

Rate rises

Denaturation

Figure 3.7: Effect of temperature on the rate of an enzyme-controlled reaction. The rate rises to the optimum temperature, then falls as the enzyme is denatured.

Reading the temperature curve

Below the optimum, a rise in temperature gives the molecules more kinetic energy. Enzyme and substrate collide more often, so the rate rises (about doubling for each 10 °C, up to a limit).
At the optimum temperature the rate is highest.
Above the optimum, hydrogen bonds break, the secondary and tertiary structures are disrupted and the active site loses its shape. The enzyme is denatured, so the rate falls steeply in spite of more collisions.
At the maximum temperature the enzyme is fully denatured and the reaction stops.

Human enzymes and thermophilic bacteria

All human enzymes have an optimum temperature of about 37 to 38 °C, but bacteria living in hot springs (thermophilic bacteria) may have an optimum temperature of 70 °C or higher. Such enzymes have been used in biological washing powders for high-temperature washes.

100

Temperature (^{\circ} C)

Rate

Most human enzymes

Thermophilic bacterial enzymes

Figure 3.8: Optimum temperature for human enzymes (about 37 to 38 °C) and for the enzymes of thermophilic bacteria (70 °C or higher).

Low temperature: the minimum temperature

If the temperature is reduced to near or below freezing point, enzymes are inactivated, not denatured. They regain their catalytic influence when higher temperatures are restored. The temperature at which an inactive enzyme becomes active again is called the minimum temperature.

The three temperatures of an enzyme
Term	Meaning	Effect on the enzyme
Minimum temperature	The temperature at which an inactive (cold) enzyme becomes active again	Inactivated at freezing, not damaged; recovers on warming
Optimum temperature	The temperature that promotes maximum activity (about 37 to 38 °C for human enzymes)	Highest rate
Maximum temperature	The temperature that causes denaturation	Structure and active site destroyed; activity lost

Worked example: reading a temperature experiment

Equal amounts of an enzyme and its substrate were kept at six temperatures. The rate of product formation is given below. (a) State the optimum temperature. (b) Explain the rise from 10 °C to 40 °C. (c) Explain the fall after 40 °C. (d) Tubes kept at 0 °C and at 60 °C are both brought to 37 °C. Which one will work again, and why?

Temperature (°C)	10	20	30	40	50	60
Rate (units per minute)	2	4	8	12	5	0

Temperature (°C)	Rate (units per minute)
10	2
20	4
30	8
40	12
50	5
60	0

Temperature (^{\circ} C)

Rate

Data of the worked example, plotted.

(a) The highest rate is 12 units per minute, at 40 °C, so the optimum temperature is 40 °C.
(b) From 10 °C to 30 °C the rate doubles for every 10 °C (2, 4, 8). Heat increases molecular motion, so enzyme and substrate molecules collide more often and the chance of a reaction rises. The gain from 30 °C to 40 °C is smaller because the doubling effect holds only up to a limit.
(c) Above 40 °C the hydrogen bonds of the enzyme break. The secondary and tertiary structures are disrupted, the active site loses its shape and the enzyme is denatured, so the rate falls to 5 and then to 0 at 60 °C even though collisions are more frequent.
(d) The tube at 0 °C works again: cold only inactivates an enzyme and its structure is intact. The tube at 60 °C stays inactive: its enzyme is denatured and the change is not reversed by cooling.

Optimum 40 °C; the 0 °C enzyme recovers (inactivated), the 60 °C enzyme does not (denatured).

3.3.2pHOften examined

Every enzyme functions most effectively over a particular pH range. The narrow range of pH at which the maximum rate of reaction is achieved is called the optimum pH.

Enzyme conformation is sensitive to pH changes because pH influences the charges on the amino acid side chains that are involved in maintaining the tertiary and quaternary structure of the enzyme. A slight change in the optimum pH of an enzyme causes ionisation of the amino acids of the enzyme, so the enzyme becomes inactive temporarily. On the other hand, extreme changes in pH alter the ionic charge of the acidic and basic groups of the enzyme and therefore disrupt the ionic bonding (denaturation) that helps to maintain the specific shape of the enzyme.

Figure 3.9: Effect of pH on the rate of an enzyme-controlled reaction. Pepsin works best at about pH 2, trypsin at about pH 8.

The optimum pH values of most enzymes fall in the range of pH 6 to 8, but there are exceptions. The protein-digesting enzyme pepsin is active in an acidic medium at pH 2, and trypsin is inactive at this pH but shows maximum activity in an alkaline medium at pH 8.

Some enzymes, like papain from green papaya, act both in acidic and alkaline media. Papain is a cysteine protease acquired from the latex of the papaya plant. It has been used for protecting plants against insects. The enzyme has a high optimal temperature (65 °C) and a wide pH range (5 to 8) for its activity.

Optimum pH of some enzymes
Enzyme	Optimum pH	Medium
Most enzymes	6 to 8	Near neutral
Pepsin	About 2	Acidic (stomach); trypsin is inactive here
Trypsin	About 8	Alkaline
Papain	Wide range, 5 to 8 (optimum temperature 65 °C)	Acidic and alkaline

Why pH matters

Each enzyme has side chains whose charges hold its tertiary and quaternary structure.
A slight change from the optimum pH changes the ionisation of these side chains; the enzyme becomes inactive for the time being.
An extreme change alters the ionic charge of the acidic and basic groups and breaks the ionic bonds: the enzyme is denatured.
Hence each enzyme gives a bell-shaped curve with a peak at its own optimum pH.

Worked example: naming enzymes from a pH graph

The graph shows the rate of three protein-digesting enzymes X, Y and Z at different pH values. The enzymes are pepsin, trypsin and papain. (a) Name X, Y and Z, giving the reason. (b) What will happen to Y if it passes into the stomach, where the pH is about 2? (c) Which enzyme can be used in both acidic and alkaline media?

Rate against pH for three protein-digesting enzymes X, Y and Z (own data).

(a) X has its peak at about pH 2, an acidic medium: it is pepsin. Y has its peak at about pH 8, an alkaline medium: it is trypsin. Z has a flat, wide peak from about pH 5 to 8: it is papain, which works over a wide range.
(b) At pH 2 trypsin is inactive. The acid changes the ionic charges on its side chains and disrupts its ionic bonds, so the shape of its active site is lost.
(c) Papain (Z), because it is active over the wide pH range of 5 to 8.

X = pepsin, Y = trypsin, Z = papain; trypsin is inactive at pH 2; papain works in acidic and alkaline media.

3.3.3Enzyme concentrationOften examined

Provided that the substrate concentration is maintained at a high level (unlimited availability), and other conditions such as pH and temperature are kept constant, the rate of reaction becomes directly proportional to the enzyme concentration.

If there is only one enzyme in the system, it can convert hundreds of substrates into products, but it takes more time. By increasing the concentration of enzyme, the number of active sites becomes larger and the rate of conversion of substrate into product becomes faster. Such an effect persists till the equilibrium state (when the concentration of enzyme and substrate becomes equal); after that, further increase in enzyme concentration has no effect upon the rate of reaction.

Figure 3.10: Relationship between enzyme concentration and the rate of an enzyme-controlled reaction. The rate is directly proportional to enzyme concentration while substrate is plentiful.

Reading the graph

Keep substrate in excess and keep pH and temperature constant.
Raise the enzyme concentration: there are more active sites.
More substrate molecules are converted per second, so the rate rises in a straight line.
When substrate becomes the limiting factor (enzyme and substrate become equal), more enzyme no longer raises the rate.

3.3Substrate concentrationOften examined

The book measures the rate of an enzymatic reaction by the amount of substrate changed, or the amount of product formed, in a period of time. Substrate concentration is the amount of substrate present for the enzyme to act on. For the enzyme concentration factor the book keeps the substrate concentration high (unlimited availability) so that the enzyme's active sites are not short of substrate; the other conditions, pH and temperature, are kept constant when one factor is studied.

3.4Enzyme inhibitionOften examined

The phenomenon in which an enzyme fails to catalyse a reaction is called enzyme inhibition. The molecules which react with an enzyme but are not converted into the desired products are called enzyme inhibitors.

In general, enzyme inhibition is a normal part of the regulation of enzyme activity within cells, but sometimes, when external factors cause enzyme inhibition, it may become dangerous for life. The molecules which act as inhibitors include poisons, cyanides, antibodies, anti-metabolites, penicillin, sulpha drugs and so on.

Inhibition may be competitive or non-competitive.

The two types of enzyme inhibition
Type of inhibition	Where the inhibitor binds	Learn in
Competitive	The active site, in competition with the substrate	Competitive inhibition
Non-competitive	A site other than the active site (the allosteric site)	Non-competitive inhibitors

3.4.1Competitive inhibitionOften examined

A type of enzyme inhibition in which enzyme activity is blocked by the presence of a chemical that competes with the substrate for binding to the active site is called competitive inhibition.

Usually a competitive inhibitor is structurally similar to the normal substrate and so fits into the active site. However, it is not similar enough to substitute fully for the normal substrate in the chemical reaction, so the enzyme cannot catalyse it to form reaction products.

Competitive inhibition is usually temporary, and the inhibitor eventually leaves the enzyme, so it is also called reversible inhibition. The level of inhibition depends on the relative concentrations of substrate and inhibitor, since they are competing for places in the enzyme's active sites. Therefore, if the concentration of the substrate is increased relative to the concentration of the inhibitor, the active site will usually be occupied by the substrate.

An example of an inhibitor is malonate. Succinate dehydrogenase, which catalyses the formation of fumarate from succinate, is competitively inhibited by malonate.

Succinate (substrate)

Malonate (competitive inhibitor) Own drawing of the molecules in Figure 3.11: malonate is like succinate but one \ce{CH2} group shorter, so it fits the active site but cannot be converted.

Figure 3.11: Effect of malonate as a competitive inhibitor. (a) Succinate binds, is converted and fumarate is released. (b) Malonate binds to the same active site but no product is formed.

How a competitive inhibitor works

The inhibitor has a shape similar to the substrate.
It enters the active site of the enzyme and forms an enzyme-inhibitor complex.
It cannot be converted into product, and while it occupies the site the real substrate cannot bind.
Because it eventually leaves, the effect is temporary (reversible), and a high concentration of substrate can crowd the inhibitor out.

Importance of competitive inhibitors

It supports the lock and key hypothesis.
It shows that substances which are similar to the substrate are not acted upon by enzymes.
Competitive inhibitors are used as drugs in the control of bacterial pathogens. Antibiotics known as sulphonamides are used to combat bacterial infection.

3.4.2Non-competitive inhibitorsOften examined

In non-competitive inhibition the inhibitor molecule binds to an enzyme at a site other than the active site. This other binding site of the enzyme is called the allosteric site. Non-competitive inhibitors inactivate the enzyme temporarily (reversible inhibition) or they denature the enzyme permanently (irreversible inhibition).

Figure 3.12: (a) Competitive inhibition: the inhibitor occupies the active site. (b) Non-competitive inhibition: the inhibitor binds to the allosteric site and the active site is no longer suitable for the substrate.

Reversible non-competitive inhibition

Reversible non-competitive enzyme inhibitors work not by preventing the formation of enzyme-substrate complexes, but by preventing the formation of enzyme-product complexes. So they prevent the substrate from being converted into product. Feedback inhibition is an example of reversible non-competitive enzyme inhibition.

Irreversible non-competitive inhibition

An irreversible non-competitive enzyme inhibitor destroys the enzyme by altering its shape, so that the substrate cannot bind to the active site. Examples of irreversible non-competitive inhibitors include cyanides and salts of heavy metals.

Cyanides are potent poisons of living organisms, because they can kill an organism by inhibiting cytochrome oxidase, which is essential for cellular respiration. They block the action of these enzymes by combining with iron that may be present in the prosthetic group.

Ions of heavy metals such as mercury, silver and copper (\ce{Hg^2+}, \ce{Ag+} and \ce{Cu^2+}) combine with thiol (-\mathrm{SH}) groups in the enzyme, breaking the disulphide bridges. These bridges are important in maintaining the tertiary structure. When they are broken, the enzyme becomes denatured and inactive.

How heavy-metal ions inactivate an enzyme

The heavy-metal ion meets the enzyme and combines with its -\mathrm{SH} (thiol) groups.
The disulphide bridges that hold the tertiary structure are broken.
The enzyme loses its shape, including the shape of its active site: it is denatured and inactive. This cannot be reversed.

Competitive and non-competitive inhibitors
Point	Competitive inhibitor	Non-competitive inhibitor
Binds to	The active site	The allosteric site, a site other than the active site
Structure	Similar to the substrate	Need not resemble the substrate
Effect of more substrate	Overcomes the inhibition (substrate competes for the active site)	No effect
Reversibility	Reversible (temporary)	Reversible or irreversible
Examples	Malonate, penicillin, sulphonamides	Cyanide, mercury, silver, copper

Reversible and irreversible non-competitive inhibitors
Point	Reversible non-competitive	Irreversible non-competitive
Effect on the enzyme	Inactivates it temporarily	Destroys it by altering its shape (denaturation)
Complexes	Does not stop ES complex forming; prevents EP complex formation	Substrate cannot bind to the active site
Examples	Feedback inhibition	Cyanides, salts of heavy metals (\ce{Hg^2+}, \ce{Ag+}, \ce{Cu^2+})

Worked example: comparing the effect of more substrate

An enzyme is tested with no inhibitor, with a competitive inhibitor and with a non-competitive inhibitor, at increasing substrate concentrations. The graph shows the three results. Say which curve (A, B or C) is which, and explain.

Own data for the worked example.

A rises quickest and reaches the highest level: it is the enzyme with no inhibitor.
B rises more slowly at first but at high substrate concentration it reaches the same level as A. Extra substrate crowds the competitive inhibitor out of the active sites, so B is the competitive inhibitor.
C stays lower however much substrate is added. The non-competitive inhibitor is bound to the allosteric site and has changed the enzyme; substrate cannot remove it. So C is the non-competitive inhibitor.

A: no inhibitor. B: competitive inhibitor (overcome by more substrate). C: non-competitive inhibitor (not overcome).

3.4.3Feedback inhibitionOften examined

The activity of almost every enzyme in a cell can be regulated by its product. When the activity of an enzyme is inhibited by its own product, it is called feedback inhibition. This is a type of reversible non-competitive inhibition. This phenomenon is part of a normal regulatory mechanism and usually happens during the regulation of metabolic pathways.

For example, the amino acid aspartate becomes the amino acid threonine by a sequence of five enzymatic reactions. When threonine, the end product of this pathway, is present in excess, it binds to an allosteric site on enzyme 1 of this pathway, and then the active site is no longer able to bind aspartate. When all the threonine is consumed in cellular events, the threonine molecule attached to the allosteric site is also removed, and the pathway resumes its activity once again.

Aspartate

Threonine Own drawing of the start and end molecules of the pathway in Figure 3.13.

Figure 3.13: Feedback inhibition. The end product threonine binds to the allosteric site of the first enzyme, E₁, and switches off the whole pathway.

Feedback control of the pathway

Aspartate is changed to threonine through intermediates A, B, C and D by five enzymes, E₁ to E₅.
When threonine accumulates, some of it binds to the allosteric site of E₁.
The active site of E₁ is changed and can no longer bind aspartate: the whole pathway slows down or stops.
When the cell uses up the threonine, it leaves the allosteric site, E₁ works again and the pathway resumes.

Worked example: what happens in the aspartate pathway?

In the pathway aspartate to A to B to C to D to threonine, state what happens to each of the following and why: (a) the pathway when threonine is present in excess, (b) the pathway when the cell has used up all its threonine, (c) the amounts of A, B, C and D while the pathway is switched off. Why is the inhibitor not acting on the last enzyme, E₅?

(a) Excess threonine binds to the allosteric site of E₁. The active site of E₁ cannot bind aspartate, so no A is made and the pathway stops.
(b) With no threonine left, the allosteric site is empty. E₁ binds aspartate again, so the pathway resumes.
(c) A, B, C and D are not made any more, because the first step is blocked. No raw material (aspartate) is wasted on making more threonine than the cell needs.
Reason for E₁: inhibiting the first enzyme switches off the whole pathway at its start. The inhibitor binds to an allosteric site, not to the active site, so this is non-competitive; it is reversible, because threonine leaves when it is consumed.

Excess threonine switches the pathway off at E₁; when it is used up the pathway restarts. It is reversible non-competitive inhibition.

Examples of inhibitors (book box)

Examples of inhibitors
Type	Examples
Competitive inhibitors	Antibodies, antimetabolites, penicillin, iodoacetate, malonate, CoA (at high concentration) and so on
Non-competitive inhibitors	Acetaldehyde di-isopropyl fluorophosphate (DFP, nerve gas), mercury, silver, copper, cyanide

3.5Classification of enzymesOften examined

Enzymes can be classified either on the basis of the reaction types that they catalyse or on the basis of the substrate on which the enzyme acts.

Ways of naming enzymes (book titbit)
Basis of naming	Example	Reading
Substrate + ase	Proteases, lipases	Act on proteins, lipids
Type of reaction	Oxidases, reductases	Catalyse oxidation, reduction
Substrate and reaction	DNA polymerase	Acts on DNA by forming a polymer
Substance synthesised	Rhodonase	Makes rhodonate

3.5.1Classification based upon reaction typeOften examined

A systematic nomenclature and classification of enzymes based on reaction types and reaction mechanism was given by the International Union of Biochemistry (in 1961). On that basis all enzymes have been classified into six groups: (1) oxidoreductases, (2) transferases, (3) hydrolases, (4) lyases, (5) isomerases and (6) ligases.

1. Oxidoreductases

These enzymes catalyse oxidation and reduction of their substrate and act by removing or adding electrons or \ce{H+} ions from or to the substrate. For example, cytochrome oxidase oxidises cytochrome.

2. Transferases

These enzymes catalyse the transfer of a specific functional group, other than hydrogen, from one substrate to another. The chemical group transferred in the process is not in a free state. For example, hexokinase transfers a phosphate group from ATP to glucose.

3. Hydrolases

These enzymes bring about the breakdown of large complex organic molecules into smaller ones by adding water (hydrolysis) and breaking specific covalent bonds. Examples are the proteolytic enzymes, which break down proteins into peptones and peptides, such as pepsin, renin and trypsin. Other digestive enzymes that work in the digestive tract are also examples of hydrolases.

4. Lyases

These enzymes catalyse the breakdown of specific covalent bonds and the removal of groups without hydrolysis. For example, histidine decarboxylase breaks the covalent bonds between carbon atoms in histidine, forming carbon dioxide and histamine.

5. Isomerases

These enzymes bring about an intra-molecular rearrangement of atoms in the molecules and thus form one isomer from another. For example, phosphohexose isomerase changes glucose 6-phosphate to fructose 6-phosphate.

\text{Glucose 6-phosphate}\ \xrightarrow{\ \text{Phosphohexose isomerase}\ }\ \text{Fructose 6-phosphate}

6. Ligases (synthetases)

These enzymes bring about the joining together of two molecules. The energy is derived by the hydrolysis of ATP. Polymerases are responsible for linking monomers into a polymer such as DNA or RNA.

The six classes of enzymes
Class	Reaction catalysed	Example from the book
1. Oxidoreductases	Oxidation and reduction: remove or add electrons or \ce{H+}	Cytochrome oxidase
2. Transferases	Transfer of a functional group (not hydrogen) from one substrate to another	Hexokinase (phosphate from ATP to glucose)
3. Hydrolases	Break large molecules into smaller ones by adding water	Pepsin, renin, trypsin
4. Lyases	Break covalent bonds and remove groups without hydrolysis	Histidine decarboxylase
5. Isomerases	Intra-molecular rearrangement to form an isomer	Phosphohexose isomerase
6. Ligases (synthetases)	Join two molecules, using energy from ATP hydrolysis	Polymerases

Worked example: classify the enzyme by its reaction

Name the class of each enzyme from what it does, and give the reason: (a) the enzyme that takes the phosphate group of ATP and attaches it to glucose, (b) the enzyme that changes glucose 6-phosphate into its isomer fructose 6-phosphate, (c) the enzyme that removes the carboxyl group of histidine as carbon dioxide without using water, (d) the enzyme that joins nucleotides into a DNA chain using ATP, (e) the enzyme that adds water to split proteins into peptides.

Part	Enzyme in the book	Class	Reason
(a)	Hexokinase	Transferase	A phosphate group is moved from one substrate (ATP) to another (glucose)
(b)	Phosphohexose isomerase	Isomerase	Atoms are rearranged inside the molecule to give an isomer
(c)	Histidine decarboxylase	Lyase	A covalent bond is broken and a group removed without hydrolysis
(d)	Polymerase	Ligase	Two molecules are joined and the energy comes from hydrolysis of ATP
(e)	Pepsin, trypsin	Hydrolase	Water is added to break covalent bonds of a large molecule

Look at the key action in each question: move a group, rearrange atoms, split without water, join using ATP, split using water.
Match the action with the class: transferase, isomerase, lyase, ligase, hydrolase.
Quote the book's example to support the answer.

(a) transferase, (b) isomerase, (c) lyase, (d) ligase, (e) hydrolase.

3.5.2Classification based upon substrateOften examined

Enzymes can be classified on the basis of the substrates they use. Some of the examples are proteases, lipases, carbohydrases and nucleases.

1. Proteases

These enzymes act upon proteins. Examples are pepsin and trypsin (both digest large polypeptides into small polypeptides or peptones), aminopeptidases and carboxypeptidases (both digest peptones into dipeptides) and erypsin (digests dipeptides into amino acids).

Own diagram: the step-by-step digestion of a protein by the proteases named in the book.

2. Lipases

These enzymes hydrolyse lipids into fatty acids and glycerol. Examples are the pancreatic lipases.

3. Carbohydrases

These enzymes cause the breakdown of carbohydrates. Examples are:

Carbohydrases
Enzyme	Substrate	Product
Amylase	Starch or glycogen	Maltose
Cellulase	Cellulose	Cellobiose, a disaccharide
Maltase	Maltose	Glucose
Sucrase	Sucrose	Glucose and fructose
Lactase	Lactose	Galactose and glucose

4. Nucleases

These are involved in the breakdown of DNA and RNA. Examples are:

Nucleases
Enzyme	Action
RNAases	Digest RNA into ribonucleotides
DNAases	Digest DNA into deoxyribonucleotides
ATPases	Cause hydrolysis of ATP in muscles and so on

Classification based upon substrate
Group	Acts on	Examples
Proteases	Proteins	Pepsin, trypsin, aminopeptidases, carboxypeptidases, erypsin
Lipases	Lipids	Pancreatic lipases
Carbohydrases	Carbohydrates	Amylase, cellulase, maltase, sucrase, lactase
Nucleases	DNA and RNA	RNAases, DNAases, ATPases

Diagnostic uses of enzymes (book box)

Diagnostic uses of enzymes
Enzyme	Use in diagnosis
Aldolase	Progressive muscular dystrophy, viral hepatitis and advanced cancer of the prostate
Creatine phosphokinase	Damage to muscle cells
Gamma-glutamyl transpeptidase	In assessing liver function
Lactic dehydrogenase	In differentiating heart attack, anaemia, lung injury or liver disease
Lipase	Damage to the pancreas

3.4Venoms as enzyme inhibitors

Snake venom is highly modified saliva that is produced by special glands of certain species of snakes. Snake venom is a combination of many toxins (proteins) and different enzymes, used for purposes like increasing the prey's uptake of toxins. Snake venom inhibits cholinesterase to make the prey lose control of its muscles. Venom is an inhibitor of cytochrome oxidase, an essential enzyme in the cells.

There are three distinct types of venom that act on the body differently:

Types of venom
Type of venom	Acts on
Hemotoxic	The heart and cardiovascular system
Neurotoxic	The nervous system and brain
Cytotoxic	Has a localised action at the site of the bite

Venom occupies the active site of the enzyme, or it combines with the iron which may be present in the prosthetic group or which may be required as an enzyme activator.

3.5STEAM activity 3.1: lactose-free milk with immobilised enzymes

Title: Enzyme engineering: creating lactose-free milk with immobilised enzymes.

Objective

To investigate the concept of immobilised enzymes and their application in the removal of lactose from milk using lactase-embedded sodium alginate beads.

Materials required

Lactase enzyme, droppers or pipettes, sodium alginate, pH meter or pH paper, calcium chloride solution, timer or stopwatch, milk (containing lactose), thermometer, beakers and stirrers, strainer or filter.

Procedure

Begin by preparing lactase-embedded sodium alginate beads, then treat the milk

Mix lactase enzyme with sodium alginate to form a homogeneous solution.
Drop small amounts of the solution into a calcium chloride bath to create beads. Allow the beads to harden.
Rinse the beads with water to remove excess calcium chloride.
Obtain fresh milk containing lactose.
Introduce the lactase-embedded beads into the milk and stir gently.
Monitor the reaction over time, noting any changes in lactose concentration.
Measure the pH of the milk before and after the enzymatic treatment.
Use a thermometer to monitor the temperature during the process.
Filter the milk to separate the beads from the liquid.
Test the resulting milk for lactose content using lactose detection strips or a lactose testing kit. As an alternative, use Benedict's test to identify the reducing sugars, such as galactose and glucose, which are produced when lactose is broken down.

Figure 3.14: Removal of lactose. (a) When small drops of the mixture enter calcium chloride solution they form beads; the alginate holds the enzyme molecules in the beads. (b) Fresh milk containing lactose is stirred gently with the alginate beads containing immobilised lactase.

Results and discussion

Compare the lactose levels in the treated milk with the original milk. Discuss the immobilisation process, enzyme-substrate specificity, and the advantages of using immobilised enzymes for lactose removal. The book asks: Does the processed milk contain lactase? What potential drawbacks can there be when making lactose-free milk with non-immobilised lactase enzyme? Explore the potential applications of immobilised enzymes in various industries, such as food and beverage.

Key terms

Metabolism: The sum of all the chemical reactions going on in a cell.
Enzymes: Biological catalysts that speed up biochemical reactions without being consumed; biological polymers that catalyse biochemical reactions. They exist in the cell as colloids.
Diastase: The name given by Payen and Persoz to whatever it was in barley seed extract that digested starch.
Ribozymes: The only enzymes that consist of RNA; found in ribosomes. Peptidyl transferase, which forms the peptide bond in protein synthesis, is an example.
Active site: A specific charge-bearing, three-dimensional cavity of 3 to 12 amino acids, brought together by folding, in which the catalytic activity of an enzyme is located.
Substrate: The reactant that is to be converted into product; it attaches to the active site by non-covalent interactions.
Binding site: The part of the active site made of the amino acids that hold the substrate.
Catalytic site: The part of the active site made of the amino acids that convert the substrate into product.
Cofactor: A non-protein part of some enzymes that helps attach the substrate and takes part in catalysis; it may be an inorganic ion, a coenzyme or a prosthetic group.
Holoenzyme: An active enzyme that needs a cofactor and has it combined with it.
Apoenzyme: The protein part of an enzyme that is catalytically inactive because the cofactor is not available.
Pepsinogen: The inactive form of pepsin secreted by the gastric glands, with an extra polypeptide fragment blocking its active site.
Pepsin: The active form of the enzyme, formed when HCl removes the extra polypeptide fragment from pepsinogen.
Activators: Detachable inorganic cofactors, metallic ions such as \ce{Fe^2+}, \ce{Mg^2+}, \ce{Cu^2+} and \ce{Zn^2+}, attached only when the substrate binds.
Coenzymes: Detachable organic cofactors, derivatives of vitamins, for example ATP, NAD^+ and FAD.
Prosthetic group: A covalently bonded organic part of an enzyme, permanently attached and not detached after the reaction; for example the iron-containing porphyrin ring of cytochromes.
Enzyme-substrate (ES) complex: The complex formed when the substrate binds to the active site of the enzyme.
Enzyme-product (EP) complex: The complex in which the substrate has been converted into product while still attached to the enzyme.
Lock and key model: Fischer's model (1894): the enzyme is a rigid lock and the substrate is a key with a complementary shape that fits the active site exactly.
Non-regulatory enzymes: Enzymes that work according to the lock and key model, for example sucrase and maltase.
Absolute specificity: The ability of an enzyme to catalyse only one substrate and essentially no others, for example urease.
Induced fit model: Koshland's model (1959): the active site is flexible and is moulded into a precise shape as the substrate interacts with the enzyme.
Regulatory (allosteric) enzymes: Enzymes that follow the induced fit mechanism, for example hexokinase.
Energy of activation: The energy that must be added to cause molecules to react with one another; the energy required to break existing bonds and begin a reaction. Enzymes lower it.
Optimum temperature: The temperature that promotes maximum enzyme activity (about 37 to 38 °C for human enzymes).
Denaturation: Disruption of the secondary and tertiary structures of an enzyme, so that it unfolds and the precise structure of its active site is lost.
Maximum temperature: The temperature that causes denaturation of the enzyme.
Minimum temperature: The temperature at which an enzyme inactivated by cold becomes active again.
Thermophilic bacteria: Bacteria living in hot springs whose enzymes may have an optimum temperature of 70 °C or higher.
Optimum pH: The narrow range of pH at which the maximum rate of reaction is achieved.
Papain: A cysteine protease from the latex of the papaya plant, active in acidic and alkaline media (pH 5 to 8), with an optimal temperature of 65 °C.
Enzyme inhibition: The phenomenon in which an enzyme fails to catalyse a reaction.
Enzyme inhibitors: Molecules that react with an enzyme but are not converted into the desired products, for example poisons, cyanides, antibodies, anti-metabolites, penicillin and sulpha drugs.
Competitive inhibition: Inhibition in which a chemical that is structurally similar to the substrate competes with it for the active site.
Reversible inhibition: Temporary inhibition; the inhibitor eventually leaves the enzyme, as in competitive inhibition.
Malonate: A competitive inhibitor of succinate dehydrogenase, which converts succinate to fumarate.
Non-competitive inhibition: Inhibition in which the inhibitor binds to a site other than the active site.
Allosteric site: The binding site on an enzyme, other than the active site, to which a non-competitive inhibitor attaches.
Reversible non-competitive inhibition: Inhibition that inactivates an enzyme temporarily by preventing formation of the enzyme-product complex, for example feedback inhibition.
Irreversible non-competitive inhibition: Inhibition that destroys the enzyme by altering its shape so the substrate cannot bind, for example by cyanides and salts of heavy metals.
Cyanides: Potent poisons that inhibit cytochrome oxidase by combining with the iron of its prosthetic group, blocking cellular respiration.
Ions of heavy metals: Ions such as \ce{Hg^2+}, \ce{Ag+} and \ce{Cu^2+} that combine with -\mathrm{SH} groups and break disulphide bridges, denaturing the enzyme.
Feedback inhibition: Inhibition of an enzyme by its own product; a reversible non-competitive inhibition that regulates metabolic pathways.
Oxidoreductases: Enzymes that catalyse oxidation and reduction by removing or adding electrons or \ce{H+}; for example cytochrome oxidase.
Transferases: Enzymes that transfer a functional group other than hydrogen from one substrate to another; for example hexokinase.
Hydrolases: Enzymes that break large molecules into smaller ones by adding water; for example pepsin, renin and trypsin.
Lyases: Enzymes that break specific covalent bonds and remove groups without hydrolysis; for example histidine decarboxylase.
Isomerases: Enzymes that rearrange atoms within a molecule to form an isomer; for example phosphohexose isomerase.
Ligases (synthetases): Enzymes that join two molecules using energy from ATP hydrolysis; polymerases are examples.
Proteases: Enzymes that act on proteins: pepsin, trypsin, aminopeptidases, carboxypeptidases and erypsin.
Lipases: Enzymes that hydrolyse lipids into fatty acids and glycerol, for example pancreatic lipases.
Carbohydrases: Enzymes that break down carbohydrates: amylase, cellulase, maltase, sucrase and lactase.
Nucleases: Enzymes involved in the breakdown of DNA and RNA: RNAases, DNAases and ATPases.
Snake venom: Highly modified saliva containing toxins and enzymes; it inhibits enzymes such as cholinesterase and cytochrome oxidase.
Immobilised enzymes: Enzymes held in beads (for example lactase in sodium alginate) so that they can be separated from the product.

Multiple choice questionsp. 96–99

Select the correct answer.

The catalytic activity of an enzyme is restricted to its small portion called

Aactive siteAnswer
Bpassive site
Cregulation site
Dallosteric site

Why

The catalytic activity is located in the active site, a small three-dimensional cavity formed by 3 to 12 amino acids (book p. 84). The allosteric (regulation) site changes the activity of the enzyme but does not catalyse the reaction itself. 'Passive site' is not a term used by the book.

Which of the following has a coenzyme activity?

ANAD^+Answer
BCa^{++}
Cboth "a" and "b"
Dnone of them

Why

NAD^+ is a coenzyme: the book says coenzymes are derivatives of vitamins, and NAD^+ is one of its examples (p. 85).

Ca^{++} is a metal ion. Metal ions are inorganic cofactors (activators), not coenzymes, so option C is wrong.

Non-competitive inhibitors react with enzymes at:

Aactive site
Ballosteric siteAnswer
Cboth "a" and "b"
Dnone of them

Why

Non-competitive inhibitors bind the allosteric site, a site other than the active site (p. 91). Option A describes competitive inhibitors, which occupy the active site, so option C is wrong too.

Which graph shows the expected relationship between enzyme activity and substrate concentration?

AGraph (A): the rate rises to a peak and then falls
BGraph (B): the rate rises and then levels offAnswer
CGraph (C): the rate falls steadily from a high value
DGraph (D): the rate rises as a straight line from the origin

Why

As substrate concentration increases, more active sites are occupied and the rate rises more slowly until all active sites are busy. The rate then levels off at a maximum (saturation). This is graph (B).

Graph (D) is tempting because the rise is almost linear at low substrate concentration, but it cannot keep rising in a straight line. Graph (A) is the shape of the temperature curve (rise to an optimum, then fall), not of substrate concentration.

The graph shows the effect of an enzyme on a reaction. Which combination identifies X, Y and Z?

Graph for this question

AX catalysed reaction; Y uncatalysed reaction; Z activation energy
BX catalysed reaction; Y uncatalysed reaction; Z energy lost during reaction
CX uncatalysed reaction; Y catalysed reaction; Z energy gained by product
DX uncatalysed reaction; Y catalysed reaction; Z overall energy changeAnswer

Why

X has the highest peak, so it needs the largest activation energy: it is the uncatalysed reaction. Y has the lower peak, so it is the catalysed reaction. Option A reverses these two.

Z is the vertical drop from the substrate level to the lower product level. That is the change in free energy (overall energy change), which an enzyme does not alter. Option A is wrong because Z is not the activation energy (that is the height of the peak above the substrate level). Option C is wrong because the product is at a lower energy, so energy is not gained.

Note. The book prints no key. Options B and D describe the same quantity Z; D is the better answer because the book labels this difference 'change in free energy' (Fig. 3.6).

Combination of apoenzyme and coenzyme produces

Aprosthetic group
BholoenzymeAnswer
Cenzyme
Disoenzyme

Why

The apoenzyme is the inactive protein part. When the cofactor (here the coenzyme) combines with it, the active complex is the holoenzyme (p. 85). A prosthetic group is a non-protein part that is permanently attached to an enzyme; it is not the product of a combination. Option C is too general, and an isoenzyme is not formed this way.

The specificity of enzyme is due to their:

Asurface configurationAnswer
BpH
Chydrogen bonding
Dhigh molecular weight

Why

The shape (configuration) of the active site is complementary to one substrate, like a key to its lock (p. 86), so the enzyme acts only on that substrate. pH changes the activity but does not decide which substrate fits. Hydrogen bonds hold the substrate once it is in place, and molecular weight does not determine specificity.

An essential feature of a competitive inhibitor is its ability to

Aactivate an operator gene
Bcombine with prosthetic group
Cmodify a substrate
Doccupy an active siteAnswer

Why

A competitive inhibitor is structurally similar to the substrate and occupies the active site, so the substrate cannot bind (p. 91). Operator genes do not belong to enzyme inhibition. The inhibitor does not combine with the prosthetic group or change the substrate.

The reaction rate of salivary amylase with starch decreases as the concentration of chloride ions is reduced. Which of the following describe the role of the chloride ions?

Aallosteric inhibitors
BcofactorsAnswer
Ccoenzyme
Dcompetitive inhibitor

Why

The rate falls when chloride is reduced, so chloride is needed for activity. Inorganic ions are the book's cofactors (activators) that are attached to the enzyme while the substrate binds (p. 85), so chloride acts as a cofactor.

Chloride is not an inhibitor, since removing it lowers the rate. It is an inorganic ion, not an organic coenzyme.

Note. The book does not state that salivary amylase needs chloride. The answer relies on the book's definition of inorganic cofactors as activators.

How does an enzyme increase the rate of a reaction?

Aby bringing the reacting molecules into precise orientationAnswer
Bby increasing the rate of random collisions of molecules
Cby shifting the point of equilibrium of the reaction
Dby supplying the energy required to start the reaction

Why

An enzyme holds the reacting molecules in the correct position in its active site and lowers the energy of activation, so the reaction proceeds faster (p. 88).

Option B is wrong because enzymes do not increase random collisions. Option C is wrong because an enzyme changes the speed, not the equilibrium position (see question 15). Option D is wrong because an enzyme lowers the energy needed; it does not supply it.

Many enzymes are secreted in inactive form to protect

Acell proteinsAnswer
Bmitochondria
Ccell membrane
Dcell DNA

Why

Enzymes that digest proteins are made as inactive precursors (such as pepsinogen, which becomes pepsin only in the stomach cavity, p. 85). This stops them digesting the proteins of the cells that make them. Mitochondria, membrane and DNA are not what the book says these enzymes protect.

Erypsin is an example of?

Acarbohydrases
BproteasesAnswer
Clipases
Dnucleases

Why

Erypsin digests dipeptides into amino acids (p. 94), so it acts on proteins and is a protease. Carbohydrases act on sugars such as starch and lactose, lipases on fats, and nucleases on DNA, RNA and ATP.

Ribozymes consist of:

Aonly protein
Bprotein + none protein part
Conly RNAAnswer
Dnone of them

Why

Ribozymes are the only enzymes that consist of RNA; they are not proteins (p. 84). Option B describes an ordinary enzyme with a cofactor, not a ribozyme.

If an enzyme solution is saturated with substrate, the most effective way to obtain an even faster yield of products is to:

Aadd more of the enzymeAnswer
Badd more substrate
Cadd an allosteric inhibitor
Dadd a noncompetitive inhibitor

Why

When the substrate is saturating, every active site is already busy, so the substrate is not the limit. The rate depends on the number of active sites, so adding more enzyme increases the rate (p. 90, enzyme concentration). Adding more substrate has no effect, and both inhibitors slow the reaction.

If an enzyme is added to a solution where its substrate and products are in equilibrium, what would occur?

ANothing: the reaction would stay at equilibriumAnswer
BAdditional products would be formed
CAdditional substrate would be formed
DThe free energy of the system would change

Why

An enzyme speeds up the forward and reverse reactions equally, so it reaches the same equilibrium sooner but does not move it. Options B and C are therefore wrong. Option D is wrong because an enzyme does not change the free energy change of a reaction (Fig. 3.6).

Enzymes

Amake it possible for cells to escape the need for energy
Bare non-protein molecules that help coenzyme
Care not affected by change of pH
Dlower the energy of activationAnswer

Why

Enzymes lower the energy of activation of a reaction (p. 88). Option A is wrong because enzymes speed reactions but do not remove the need for energy. Option B is wrong because most enzymes are globular proteins, not non-protein helpers. Option C is wrong because enzyme activity depends on pH (p. 89).

An allosteric site on an enzyme on an enzyme is

AThe same as the active site
BWhere ATP attaches and gives up its energy
COften involved in feedback inhibitionAnswer
DAll of these are correct

Why

An allosteric site is a site separate from the active site (p. 91). It binds an end product or other regulator, so it is often involved in feedback inhibition (p. 92). Option A is wrong because it is a different site, and option B is wrong because an allosteric site does not release energy from ATP. Option D is therefore wrong.

At high temperature

Acan affect the shape of an enzymeAnswer
Blowers the energy of activation
Cmakes cells less susceptible to diseases
Dboth A and C are correct

Why

High temperature breaks the hydrogen bonds that keep the secondary and tertiary structure, so the enzyme unfolds and is denatured (p. 89). Option B is wrong because heat raises molecular motion; it does not lower the energy of activation. Option C is not supported by the book, so option D is wrong.

The coenzyme is:

Aoften a metal
Balways a protein
Coften a vitaminAnswer
Dalways an inorganic compound

Why

Coenzymes are organic molecules that are derivatives of vitamins (p. 85), so the best answer is C. Metals are inorganic cofactors, not coenzymes. A coenzyme is never a protein, and it is not an inorganic compound.

Blocking of enzyme action by blocking its active site is called as:

Afeedback inhibition
Ballosteric inhibition
Ccompetitive inhibitionAnswer
Dnon-competitive inhibition

Why

A competitive inhibitor occupies the active site and so blocks the substrate from binding (p. 91). Non-competitive inhibitors, including allosteric and feedback inhibition, act at a site other than the active site.

Short answer questionsp. 99

Answer each question in three clear points.

13 marks

What are ribozymes?

Solution

Ribozymes are enzymes made of RNA rather than protein. They are the only enzymes that consist of RNA.

They are found in ribosomes, the sites of protein synthesis.

Example: peptidyl transferase, a ribozyme that forms the peptide bond between amino acids during protein synthesis (p. 84).

23 marks

What is the structure of enzyme?

Solution

Enzymes are globular proteins made of one or more polypeptide chains. Each chain is a linear sequence of amino acids that folds into a three-dimensional structure.

The folding is set by secondary and tertiary structure, which hold the enzyme in its precise shape.

The catalytic activity is located in the active site, a small three-dimensional cavity of 3 to 12 amino acids. It has a binding site (holds the substrate) and a catalytic site (converts it into product).

33 marks

Explain the enzyme pepsin which does not require cofactor.

Solution

Pepsin is secreted by the gastric glands in an inactive form called pepsinogen. Pepsinogen has an extra polypeptide fragment attached to its active site, which stops the substrate from binding.

When pepsinogen meets the hydrochloric acid of the stomach cavity, the extra fragment is removed and the active site is freed.

The enzyme is then active as pepsin, so it needs no cofactor. Pepsin is a protein-digesting enzyme that acts at pH 2.

Note. The book calls inactive pepsinogen an apoenzyme, although no cofactor is involved. Activation here is by removing a polypeptide fragment, not by adding a cofactor.

43 marks

What is prosthetic group? Give an example.

Solution

A prosthetic group is a non-protein part of an enzyme that is covalently bonded to the enzyme.

It is permanently attached and does not detach after the reaction is complete, unlike a coenzyme.

Example: the iron-containing porphyrin ring attached to enzymes such as cytochromes (p. 85).

53 marks

What is the mechanism of enzyme action?

Solution

The substrate binds to the active site, forming the enzyme-substrate (ES) complex: \mathrm{E + S \rightarrow ES}.

While it is attached to the enzyme, the substrate is converted into product, forming the enzyme-product (EP) complex: \mathrm{ES \rightarrow EP}.

The product is released and the enzyme is unchanged and free to start again: \mathrm{EP \rightarrow E + P} (p. 86).

63 marks

What is the role of free energy of activation in a chemical reaction?

Solution

The energy of activation is the energy that must be supplied to bring reactant molecules into a reactive state, so that bonds can break and new bonds form. Only molecules with this energy react when they collide.

The higher the energy of activation, the fewer molecules have enough energy, so the slower the reaction.

An enzyme lowers the energy of activation, which increases the rate. It does not change the free energy change of the reaction (p. 88).

73 marks

List the external conditions which affect rate of enzyme reaction.

Solution

Temperature: the rate rises to an optimum and then falls as the enzyme denatures.
pH: each enzyme works best in its optimum pH range; extreme pH disturbs the ionic bonds.
Enzyme concentration: with substrate plentiful, the rate is directly proportional to enzyme concentration.
Substrate concentration: the rate rises and then levels off when all active sites are occupied.

83 marks

Compare the optimum temperatures of enzymes of human and thermophilic bacteria.

Solution

Feature	Human enzymes	Enzymes of thermophilic bacteria
Optimum temperature	About 37-38^\circ\mathrm{C}	70^\circ\mathrm{C} or higher
Effect of higher temperature	Denatured: secondary and tertiary structure is disrupted	Stay active at temperatures that would denature human enzymes
Where found or used	Human body	Hot springs; used in high temperature washing powders

So the optimum temperature of thermophilic bacterial enzymes is about 30^\circ\mathrm{C} higher than that of human enzymes (p. 89).

93 marks

Describe the range of pH at which human enzymes function.

Solution

Most human enzymes have an optimum pH in the range 6 to 8, close to neutral.

Some enzymes work outside this range: pepsin is active at about pH 2 in the stomach, and trypsin is active at about pH 8 in alkaline medium.

Outside its optimum range, a change of pH alters the ionisation of the amino acid side chains. This disrupts the ionic bonds that keep the enzyme in shape, so the enzyme is denatured (p. 89 to 90).

Note. The book gives pH 6 to 8 for most enzymes and names pepsin and trypsin as exceptions. The answer follows that wording.

103 marks

What are enzyme inhibitors? Name the molecules which act as enzyme inhibitors.

Solution

Enzyme inhibitors are molecules that react with an enzyme but are not converted into the desired product, so the enzyme fails to catalyse the reaction (p. 90).

Inhibition is a normal part of regulation inside cells, but external factors can make it dangerous to life.

Molecules that act as inhibitors include poisons, cyanides, antibodies, antimetabolites, penicillin, iodoacetate, malonate, CoA (in high concentration), sulpha drugs, acetaldehyde, DFP (nerve gas), and ions of mercury, silver and copper (p. 91 to 93).

113 marks

What is the importance of competitive enzyme inhibitors?

Solution

They support the lock and key hypothesis: a competitive inhibitor fits the active site, showing that the active site has a specific shape.

They show that substances similar to the substrate are not acted upon by the enzyme, because they cannot be converted into product.

They are used as drugs to control bacterial pathogens. Sulphonamides, for example, are antibiotics that combat bacterial infection (p. 91).

123 marks

Describe cyanides as irreversible non-competitive inhibitor.

Solution

Cyanide binds to the iron of the prosthetic group of the enzyme cytochrome oxidase, not to the active site, so it is non-competitive. The enzyme's shape is changed and the substrate can no longer bind.

The binding is permanent: the enzyme is destroyed or denatured, so the inhibition is irreversible.

Cytochrome oxidase is essential for cellular respiration, so cyanide blocks respiration and can kill the organism (p. 92).

133 marks

Describe ions of heavy metals as irreversible non-competitive inhibitor.

Solution

Ions of heavy metals such as mercury (\mathrm{Hg^{2+}}), silver (\mathrm{Ag^{+}}) and copper (\mathrm{Cu^{2+}}) bind to thiol (-\mathrm{SH}) groups of the enzyme, away from the active site, so they act as non-competitive inhibitors.

They break the disulphide bridges that maintain the tertiary structure of the enzyme, so its shape is altered and the substrate cannot bind.

The bridges cannot re-form, so the enzyme becomes denatured and permanently inactive. This is why the inhibition is irreversible (p. 92).

14(a)3 marks

Write the difference between: binding site and catalytic site of an enzyme.

Solution

Feature	Binding site	Catalytic site
Function	Holds the substrate in position	Converts the substrate into product
Type of bonds	Non-covalent bonds: hydrogen and hydrophobic bonds	Acts on the bonds of the substrate, breaking or forming them
Role in specificity	Decides which substrate can bind	Decides which reaction is catalysed

14(b)3 marks

Write the difference between: apoenzyme and holoenzyme.

Solution

Feature	Apoenzyme	Holoenzyme
Composition	Protein part of the enzyme alone	Apoenzyme with its cofactor attached
Activity	Inactive (catalytically inactive)	Active
Formation	Before the cofactor binds	When the cofactor combines with the apoenzyme

14(c)3 marks

Write the difference between: prosthetic group and coenzyme.

Solution

Feature	Prosthetic group	Coenzyme
Attachment	Covalently bonded and permanently attached	Loosely attached; detaches after the reaction
Nature	Non-protein part, such as an iron porphyrin ring	Organic molecule derived from vitamins
Examples	Iron-containing porphyrin ring of cytochromes	NAD^+, FAD, ATP (book examples)

14(d)3 marks

Write the difference between: inorganic cofactor and organic cofactor.

Solution

Feature	Inorganic cofactor	Organic cofactor
Nature	Metal ions, such as \mathrm{Fe^{2+}}, \mathrm{Mg^{2+}}, \mathrm{Cu^{2+}}, \mathrm{Zn^{2+}}	Organic molecules: coenzymes and prosthetic groups
Attachment	Detachable; attached while the substrate binds (activators)	Coenzymes are detachable; prosthetic groups are permanent
Origin	Inorganic ions	Derivatives of vitamins (coenzymes) or organic rings (prosthetic groups)

14(e)3 marks

Write the difference between: lock and key model and induced fit model of enzyme action.

Solution

Feature	Lock and key model	Induced fit model
Proposed by	Emil Fischer (1894)	Koshland (1959)
Active site	Rigid; its shape does not change	Flexible; it is moulded round the substrate
Fit with substrate	Complementary to one substrate, like a key to its lock	Changes shape on binding, so related substrates can also fit

14(f)3 marks

Write the difference between: competitive and noncompetitive enzyme inhibitors.

Solution

Feature	Competitive inhibitor	Non-competitive inhibitor
Binding site	Active site	Allosteric site (not the active site)
Shape	Similar to the substrate	Not similar to the substrate
Effect of more substrate	Can be overcome: more substrate displaces the inhibitor	Not overcome by more substrate

14(g)3 marks

Write the difference between: reversible non-competitive enzyme inhibitors and irreversible non-competitive enzyme inhibitors.

Solution

Feature	Reversible non-competitive inhibitor	Irreversible non-competitive inhibitor
Effect on enzyme	Inactivates it temporarily; the enzyme regains its shape when the inhibitor leaves	Destroys the enzyme by altering its shape permanently
Example	Feedback inhibition, where the end product binds the allosteric site	Cyanide; ions of mercury, silver and copper
Recovery	Activity returns when the inhibitor is removed	No recovery; the enzyme is denatured

Extensive answer questionsp. 100

18 marks

Write the properties of enzymes.

Solution

Definition

Enzymes are biological catalysts. They speed up biochemical reactions without being used up, and they exist in the cell as colloids.

Properties

Globular proteins: enzymes are globular proteins made of one or more polypeptides. The exception is the ribozymes, which consist of RNA.
Active site: the catalytic activity is located in a small three-dimensional cavity, the active site, so only a small part of the molecule does the catalysis.
Specificity: each enzyme acts on a particular substrate or a particular type of reaction, because the shape of its active site fits only certain substrates.
Lower energy of activation: enzymes lower the energy of activation but do not change the free energy change of the reaction.
Temperature: activity rises with temperature up to an optimum, which for most human enzymes is about 37-38^\circ\mathrm{C}. Above it the enzyme denatures and loses activity.
pH: each enzyme has an optimum pH range. A change in pH alters the charges on the amino acids and disturbs the ionic bonds that keep the enzyme in shape.
Active and inactive forms: some enzymes are secreted in an inactive form, such as pepsinogen, and are activated only where they are needed.
Inhibition: enzyme activity can be blocked by inhibitors, which is part of normal regulation inside the cell.
Action outside the cell: enzymes keep their catalytic action when extracted from cells, so they can act in vitro.

Summary

Enzymes are efficient, specific, sensitive to temperature and pH, and regulated by inhibitors. Their properties all follow from the shape of their active site.

28 marks

Explain the role and component parts of the active site of an enzyme.

Solution

The active site

The active site is the small part of an enzyme where its catalytic activity is located. It is a specific three-dimensional cavity formed by 3 to 12 amino acids that are brought together by the secondary and tertiary folding of the polypeptide, even though they may be far apart in the chain. For example, the active site of aldolase consists of glycine, histidine and alanine.

Component parts

Binding site: the amino acids that attach the substrate to the enzyme by non-covalent bonds, such as hydrogen bonds and hydrophobic interactions. They hold the substrate in the correct place and decide which substrate can bind.
Catalytic site: the amino acids that act on the bound substrate and convert it into product, by breaking or forming its bonds.

The active site has a binding site, which holds the substrate, and a catalytic site, which converts it into product.

Role of the active site

The binding site holds the substrate, so that the enzyme-substrate (ES) complex forms.
The catalytic site brings the reacting groups together and changes the bonds of the substrate. This lowers the energy of activation.
The product is released, the active site is free again, and the same site can be used for another reaction.

Because the shape of the active site is designed for the substrate, only a particular substrate can bind, although some related substrates can also fit.

38 marks

What are cofactors? Describe the two types of cofactors by giving examples.

Solution

What cofactors are

Cofactors are non-protein parts that some enzymes need in order to be active. A cofactor helps to attach the substrate to the active site and also takes part in the catalytic process. The final shape of the active site is set only after the cofactor is attached.

The two types

The question asks for two types, so the cofactors are grouped as inorganic cofactors and organic cofactors. Organic cofactors are then divided into coenzymes and prosthetic groups.

Type	Nature	Attachment	Examples
Inorganic cofactor (activator)	Metal ions	Detachable: attached only while the substrate binds	\mathrm{Fe^{2+}}, \mathrm{Mg^{2+}}, \mathrm{Cu^{2+}}, \mathrm{Zn^{2+}}
Organic cofactor: coenzyme	Organic molecule derived from vitamins	Detachable	ATP, NAD^+, FAD (book examples)
Organic cofactor: prosthetic group	Non-protein organic part	Covalently bonded and permanently attached	Iron-containing porphyrin ring of cytochromes

Holoenzyme and apoenzyme

An enzyme that needs a cofactor is inactive as a protein alone. The protein part is the apoenzyme. When the cofactor combines with it, the active enzyme is the holoenzyme.

Apoenzyme plus cofactor gives the active holoenzyme.

Example: hexokinase needs \mathrm{Mg^{2+}} to convert glucose and ATP into glucose 6-phosphate and ADP, as the book shows (p. 85).

Note. The question asks for two types of cofactors, but the book describes three: inorganic ions, organic molecules (coenzymes) and the prosthetic group. The answer covers all three and groups them as inorganic and organic.

48 marks

Explain the mechanism of enzyme action through induced fit model.

Solution

The induced fit model

Koshland proposed the induced fit model in 1959. According to this model the active site is flexible rather than rigid. Its amino acids are moulded into a precise shape only when the substrate binds, and this change of shape is what converts the substrate into product.

Mechanism in steps

The enzyme has a flexible active site that is not yet exactly complementary to the substrate.
The substrate approaches and binds to the active site by non-covalent bonds. This starts the change of shape in the enzyme.
The active site changes its shape and its amino acids reposition around the substrate, forming a precise fit: this is the enzyme-substrate complex. The reacting groups are brought together and the substrate is converted into product.
The product is released and the active site regains its original shape, so the enzyme is ready to catalyse a new reaction.

Induced fit model: the active site changes shape around the substrate and returns to its original shape after the products are released.

Significance

A flexible active site allows an enzyme to fit more than one closely related substrate, so it can catalyse more than one type of related reaction.
Enzymes that work by induced fit are called regulatory or allosteric enzymes. Hexokinase is the book's example.

Note. The book's equation on p. 87 shows carbonic anhydrase adding O_2 to haemoglobin. That is a misprint: its correct action is the second equation, CO_2 + H_2O to H_2CO_3. The answer uses hexokinase instead.

58 marks

Explain the mechanism of enzyme action through lock and key model.

Solution

The lock and key model

Emil Fischer proposed the lock and key model in 1894. The enzyme is the lock and the substrate is the key. The active site is a rigid, notched region whose shape is complementary to the substrate, just as the notch of a key fits its lock.

Mechanism in steps

The substrate (key) has a shape similar to the active site (lock).
The substrate binds tightly to the active site by non-covalent bonds, forming the enzyme-substrate complex.
While it is attached, the substrate is converted into product, forming the enzyme-product complex.
The product leaves and the active site is free again. The enzyme itself is unchanged and can act on another substrate.

Lock and key model: the substrate fits the rigid active site exactly, and the enzyme is not changed.

Significance and limitations

It explains high specificity: only a substrate of the right shape fits the active site.
It does not explain why some enzymes act on several related substrates, or why the active site can change shape. The induced fit model covers these points.
Some books reverse the names, with the key as the enzyme and the lock as the substrate. The meaning is the same.

68 marks

Explain how an enzyme catalyzes specific reactions.

Solution

Specificity of the active site

An enzyme catalyses a specific reaction because its active site has a specific shape and a specific arrangement of amino acids. Only a substrate that fits the binding site is held, and only the bond that lies next to the catalytic site is changed.

Steps

The binding site has amino acids that attach to the substrate by hydrogen and hydrophobic bonds. Only a substrate of complementary shape binds.
The substrate is held in the correct orientation, so that the catalytic site can act on the bond that must be broken or formed.
The catalytic site converts the substrate into product and lowers the energy of activation, so the reaction proceeds quickly.
The product is released and the enzyme can catalyse the next reaction of the same kind.

Only substrate A fits the active site. Substrates B and C do not.

Examples

Urease acts only on urea, so it shows absolute specificity (book p. 87).
Sucrase and maltase each catalyse one specific reaction, and are the book's examples of enzymes that work by the lock and key model.
Enzymes with relative specificity act on a group of related substrates, as explained by the induced fit model.

78 marks

Explain through graph how an enzyme speeds up reaction by lowering the energy of activation.

Solution

Energy of activation

Molecules must gain a minimum energy, the energy of activation, before they can react. Without an enzyme this energy is high, so only a few molecules react at a time. An enzyme provides a different pathway with a lower energy barrier.

Energy profile of an enzyme-controlled reaction: the enzyme lowers the activation energy, but the energy of the reactants and products, and so the free energy change, is the same.

How the graph explains the speed-up

Reactants start at the same energy level with or without the enzyme.
Without the enzyme, the reactants must climb a high peak (the transition state). The activation energy is the height from the reactant level to the top of this peak.
With the enzyme, the reaction follows a pathway through the enzyme-substrate complex with a lower peak, so the activation energy is smaller.
More molecules have enough energy to cross the lower barrier in a given time, so the rate of reaction increases.
The products are at the same level as before, so the change in free energy is unchanged. The enzyme speeds the reaction but does not change the overall energy change.

88 marks

Describe the effect of temperature on the rate of enzyme action.

Solution

Rise in rate

Heating increases molecular motion, so the substrate and enzyme molecules collide more often. Over a range of temperature, an increase of 10^\circ\mathrm{C} roughly doubles the rate of reaction.

Effect of temperature on the rate of an enzyme-controlled reaction

The rate doubles for every 10^\circ\mathrm{C} rise up to the optimum, then falls as the enzyme denatures.

Above the optimum

The rate reaches a maximum at the optimum temperature. For human enzymes this is about 37-38^\circ\mathrm{C}.
Above the optimum the secondary and tertiary structures are disrupted, mainly by breaking hydrogen bonds. The enzyme unfolds and the precise shape of the active site is lost. This is denaturation.
The rate then falls quickly and the enzyme becomes inactive. The temperature that causes denaturation is the maximum temperature.

Low temperature and other organisms

Near or below freezing point the enzyme is inactivated, but it is not denatured, so its activity returns when the temperature rises again. This is the minimum temperature. Enzymes of thermophilic bacteria in hot springs have an optimum of 70^\circ\mathrm{C} or higher, which is why they are used in high temperature washing powders.

98 marks

Describe how the concentration of enzyme affects the rate of enzyme action.

Solution

Direct proportion

If the substrate is available in plenty and the temperature and pH are kept constant, the rate of reaction is directly proportional to the enzyme concentration.

Explanation in steps

Each enzyme molecule has its own active site. More enzyme molecules mean more active sites available at any moment.
More active sites convert more substrate into product in the same time, so the rate increases in proportion to the enzyme concentration.
A single enzyme can convert many substrate molecules, but it takes longer; more enzyme molecules shorten the time.

Effect of enzyme concentration on the rate of reaction

With substrate plentiful, the rate is a straight line through the origin (book Fig. 3.10).

The straight-line relation holds only while the substrate is not used up. When the substrate becomes the limiting factor, adding more enzyme no longer increases the rate in the same way.

Note. The book says the effect stops when the enzyme and substrate concentrations become equal. The plateau actually occurs when the substrate becomes limiting, so the answer follows that reasoning.

108 marks

Explain the effect of substrate concentration on the rate of enzyme action.

Solution

Effect

With the enzyme concentration, temperature and pH kept constant, the rate increases as the substrate concentration increases, because more substrate molecules collide with the active sites.

Explanation in steps

At low substrate concentration many active sites are empty, so the rate rises as substrate is added.
As the substrate concentration rises, more active sites are occupied at any moment, so the rate rises more slowly.
At high substrate concentration nearly all active sites are occupied. The enzyme works at its maximum speed, and adding more substrate has no further effect. This is saturation.

Effect of substrate concentration on the rate of an enzyme-controlled reaction

The rate rises, then levels off at a maximum set by the amount of enzyme present.

So the rate rises and then levels off. The level of the plateau depends on the amount of enzyme, not on more substrate.

118 marks

Describe enzymatic inhibition, its types and its significance.

Solution

Enzyme inhibition

Enzyme inhibition is the phenomenon in which an enzyme fails to catalyse a reaction. The molecules that react with the enzyme but are not converted into the desired product are called enzyme inhibitors. Inhibition is a normal part of regulation inside cells, but external factors can make it dangerous to life.

Types

Feature	Competitive inhibition	Non-competitive inhibition
Binding site	Active site	Allosteric site (a site other than the active site)
Shape of inhibitor	Similar to the substrate	Not similar to the substrate
Effect on enzyme	Blocks the active site, so the substrate cannot bind	Changes the shape of the active site, so the substrate cannot bind
Reversibility	Usually reversible (temporary)	Reversible (feedback inhibition) or irreversible (cyanide, heavy metal ions)
Effect of more substrate	Can be overcome: more substrate displaces the inhibitor	Not overcome by more substrate
Example	Malonate, which blocks succinate dehydrogenase	Cyanide; ions of mercury, silver and copper

Competitive inhibitors block the active site. Non-competitive inhibitors bind the allosteric site and change the shape of the active site.

Significance

Regulation: feedback inhibition controls metabolic pathways by stopping an enzyme when its end product is in excess (question 12).
Drugs: competitive inhibitors such as sulphonamides and penicillin block enzymes of bacteria and are used to control bacterial pathogens.
Poisons: cyanide and heavy metal ions irreversibly inhibit enzymes such as cytochrome oxidase, which stops cellular respiration.
Lock and key: competitive inhibitors show that molecules similar to the substrate are not acted upon by the enzyme.

128 marks

Explain feedback mechanism with reference to enzymes.

Solution

The feedback mechanism

The activity of almost every enzyme in a cell can be regulated by its own product. When the product inhibits an enzyme of the pathway that makes it, the process is called feedback inhibition. It is a type of reversible non-competitive inhibition and is part of the normal regulation of metabolic pathways.

Example: threonine

The amino acid aspartate is converted into threonine by a sequence of five enzymatic reactions, E1 to E5, with the intermediates A, B, C and D.

Feedback inhibition: threonine, the end product, switches off the first enzyme, E1, by binding its allosteric site.

Steps

Threonine is made in excess and accumulates in the cell.
Threonine binds to the allosteric site of enzyme E1, the first enzyme of the pathway.
The shape of the active site of E1 changes, so it can no longer bind aspartate.
The pathway stops, so no further intermediates are made.
When the threonine is used up in cellular events, it leaves the allosteric site and the pathway resumes its activity.

Significance

It prevents wasteful production of an end product that is already in excess.
It keeps the level of each metabolite in balance with the needs of the cell.
Unlike the irreversible inhibition by cyanide, it is reversible, because the threonine leaves the allosteric site.

138 marks

Classify enzymes on the basis of reactions catalyzed.

Solution

In 1961 the International Union of Biochemistry classified enzymes into six groups on the basis of the reaction types they catalyse and their mechanism.

Group	Reaction catalysed	Example
Oxidoreductases	Oxidation or reduction: remove or add electrons or H^+ ions to the substrate	Cytochrome oxidase oxidizes cytochrome
Transferases	Transfer of a functional group other than hydrogen from one substrate to another; the group is not free	Hexokinase transfers a phosphate group from ATP to glucose
Hydrolases	Breakdown of large molecules into smaller ones by adding water (hydrolysis)	Pepsin, renin and trypsin break proteins into peptides
Lyases	Breakdown of covalent bonds and removal of groups without hydrolysis	Histidine decarboxylase forms carbon dioxide and histamine from histidine
Isomerases	Intramolecular rearrangement of atoms, forming one isomer from another	Phosphohexose isomerase changes glucose 6-phosphate to fructose 6-phosphate
Ligases (synthetases)	Joining of two molecules, with energy derived from hydrolysis of ATP	Polymerases link monomers into DNA or RNA

142026 Annual I, Set B, Q.2 (ii)8 marks

Classify enzymes on the basis of the substrate they use.

Solution

Enzymes can also be classified on the basis of the substrate they act upon. Classification by reaction says what the enzyme does; classification by substrate says what it acts on.

Class	Substrate	Products	Examples
Proteases	Proteins and peptides	Small polypeptides, peptones, dipeptides or amino acids	Pepsin, trypsin, aminopeptidases, carboxypeptidases, erypsin
Lipases	Lipids	Fatty acids and glycerol	Pancreatic lipases
Carbohydrases	Carbohydrates	Disaccharides or monosaccharides	Amylase (starch to maltose), cellulase, maltase, sucrase, lactase
Nucleases	DNA, RNA and ATP	Ribonucleotides or deoxyribonucleotides; ATP is hydrolysed	RNAases, DNAases, ATPases

FBISE 2025 and 2026 questions

Questions from the FBISE HSSC-I 2026 (1st Annual) and FBISE HSSC-I 2025 (1st Annual) papers that belong to this unit, answered the way an examiner expects.

2026 Annual I, Set B, MCQ 41 mark

The minimum energy required to start a chemical reaction is called:

APotential energy
BActivation energyAnswer
CKinetic energy
DHeat energy

Why

The energy that must be added to make molecules react is the energy of activation. In the book it is the energy needed to break the existing bonds and begin the reaction; an enzyme lowers it. Potential energy is energy stored in a system and kinetic energy is energy of motion, so neither names the starting energy of a reaction. Heat is only the way the energy of activation is supplied in a non-living system, not the name of the energy itself.

2025 Annual I, Set B, MCQ 81 mark

The compound malonate is competitive inhibitor of:

AFumarate
BSuccinate
CSuccinic dehydrogenaseAnswer
DHistidine decarboxylase

Why

Malonate has a shape similar to succinate, the substrate of the enzyme succinate dehydrogenase (the book writes succinic dehydrogenase). It enters the active site, forms an enzyme-inhibitor complex and cannot be converted into product, so it competitively inhibits the enzyme. Succinate (B) is the substrate that malonate imitates, not the thing it inhibits, and fumarate (A) is the product of the reaction. Histidine decarboxylase (D) is a lyase and has nothing to do with malonate.

2025 Annual I, Set B, Q.2 (i) OR3 marks

Compare the characteristics of Cofactors of enzymes with one example of each.

Solution

A cofactor is a non-protein part that some enzymes need to become active. There are three types (1 mark each):

Point	Inorganic ions (activators)	Coenzymes	Prosthetic group
Nature	Metallic ions such as Fe, Mg, Cu, Zn	Organic molecules, derivatives of vitamins	Organic part covalently bonded to the enzyme
Attachment	Detachable; bound when the substrate binds	Detachable; bound when the substrate binds	Permanently attached; does not detach after the reaction
One example	Mg^{2+} with hexokinase	NAD^+ with alcohol dehydrogenase	Iron-containing porphyrin ring of cytochromes

2026 Annual I, Set B, Q.2 (ii)1+3 = 3 marks

Classify enzymes on the basis of the substrates they act upon, giving any three examples.

Solution

The book classifies enzymes by the substrate they act upon into four groups. Any three, each with examples, earn the marks (1 mark per group with its examples).

Class (1 mark each)	Acts on (substrate)	Products	Examples
Proteases	Proteins and peptides	Small polypeptides, peptones, dipeptides or amino acids	Pepsin, trypsin, erypsin
Lipases	Lipids	Fatty acids and glycerol	Pancreatic lipases
Carbohydrases	Carbohydrates	Disaccharides or monosaccharides	Amylase (starch to maltose), maltase, sucrase, lactase
Nucleases (fourth group)	DNA, RNA (and ATP)	Ribonucleotides or deoxyribonucleotides	RNAases, DNAases, ATPases

2026 Annual I, Set B, Q.2 (vii)1+2 = 3 marks

What is meant by minimum temperature? How it affects enzymatic action?

Solution

Meaning of minimum temperature (1 mark)

The minimum temperature is the temperature at which an enzyme that has been inactivated by cold becomes active again when it is warmed.

Effect on enzymatic action (2 marks)

Near or below freezing point enzymes are inactivated, not denatured: molecular motion is too slow for enzyme and substrate to react, but the enzyme structure is not damaged.
When the temperature is raised the activity returns, and the rate then rises (about double for every 10 degrees Celsius) up to the optimum, 37 to 38 degrees Celsius for human enzymes.

Term	Meaning	Effect on the enzyme
Minimum temperature	Temperature at which an inactive (cold) enzyme becomes active again	Inactivated at freezing, not damaged; recovers on warming
Optimum temperature	Temperature of maximum activity	Highest rate
Maximum temperature	Temperature that causes denaturation	Structure and active site destroyed

2025 Annual I, Set B, Q.2 (x)3 marks

Complete the table with reference to enzymes: [Table]

	Enzyme	Mechanism of action	Example
a	______	Break large molecules with water	______
b	Lyases	______	______
c	______	______	Cytochrome oxidase

Solution

Use the six groups of enzymes classified by reaction type. Each row is worth 1 mark.

	Enzyme	Mechanism of action	Example
a	Hydrolases	Break large molecules into smaller ones by adding water (hydrolysis)	Pepsin (also renin, trypsin)
b	Lyases	Break specific covalent bonds and remove groups without hydrolysis	Histidine decarboxylase
c	Oxidoreductases	Catalyse oxidation and reduction by removing or adding electrons or H^+ ions	Cytochrome oxidase

2026 Annual I, Set D, MCQ 41 mark

Enzymes speed up reactions by:

AIncreasing activation energy
BDecreasing activation energyAnswer
CConsuming substrates
DChanging products

Why

Enzymes are biological catalysts. They speed up a reaction by lowering the energy of activation (Figure 3.6 of the book): the reactants and products stay at the same energy levels, but the hill between them is lower. A is the opposite of what happens. An enzyme is not consumed (it comes out unchanged, so C is wrong) and it does not change what the products are (D is wrong).

2025 Annual I, Set D, MCQ 51 mark

Which enzymes break specific covalent bonds without hydrolysis?

AOxidoreductases
BHydrolases
CTransferases
DLyasesAnswer

Why

Lyases break specific covalent bonds and remove groups without hydrolysis (for example histidine decarboxylase). Hydrolases (B) break bonds too, but they do it by adding water. Oxidoreductases (A) add or remove electrons or H^+, and transferases (C) move a functional group from one substrate to another.

2026 Annual I, Set D, Q.2 (ii)1+3 = 3 marks

Write any three examples of enzymes and their specific substrates.

Solution

Each enzyme has a specific substrate, because its active site is shaped for that substrate. Any three of these (1 mark each):

Enzyme	Specific substrate	Product
Pepsin	Proteins	Small polypeptides (peptones)
Amylase	Starch or glycogen	Maltose
Lactase	Lactose	Galactose and glucose
Sucrase	Sucrose	Glucose and fructose
Pancreatic lipase	Lipids	Fatty acids and glycerol
Urease	Urea (absolute specificity)	Ammonia and carbon dioxide

2026 Annual I, Set D, Q.2 (vii)1+2 = 3 marks

What is meant by maximum temperature. How it affects enzymatic action?

Solution

Meaning of maximum temperature (1 mark)

The maximum temperature is the temperature that causes denaturation of the enzyme.

Effect on enzymatic action (2 marks)

Above the optimum (37 to 38 degrees Celsius for human enzymes) the hydrogen bonds break and the secondary and tertiary structures are disrupted. The enzyme unfolds and the precise shape of the active site is lost.
The substrate can no longer bind, so the rate falls steeply even though collisions are more frequent. At the maximum temperature the enzyme is fully denatured and the reaction stops. This cannot be reversed.

Effect of temperature on the rate of an enzyme-controlled reaction

The rate doubles for every 10^\circ\mathrm{C} rise up to the optimum, then falls as the enzyme denatures.

2025 Annual I, Set D, Q.4 OR4+3 = 7 marks

Explain mechanism of enzyme action through 'induced fit' and 'lock and key' models.

Solution

The mechanism of enzyme action is the same in both models: the enzyme (E) and substrate (S) form an enzyme-substrate complex (ES), the substrate is changed into product while attached (EP), and the product is released, leaving the enzyme unchanged. The models differ in how the active site fits the substrate.

Lock and key model

Proposed by Emil Fischer in 1894.
The enzyme is the lock and the substrate is the key. The active site has a shape complementary to the substrate, so only that substrate fits, like a key in its lock.
The enzyme is rigid: the shape of the active site does not change on binding.
It explains the high specificity of enzymes. Only a few enzymes follow it (non-regulatory enzymes), for example sucrase and maltase; urease shows absolute specificity.

Lock and key model: the substrate fits the rigid active site exactly.

Induced fit model

Proposed by Koshland in 1959.
The active site is flexible. At first it does not fit the substrate completely; the amino acids of the active site are moulded into the precise shape as the substrate binds. This change of shape makes the catalysis effective.
When the reaction is over the product leaves and the active site regains its original shape.
Because it is flexible, related substrates can bind, so one enzyme can carry out more than one related reaction. Enzymes that follow it are regulatory or allosteric enzymes, for example hexokinase; carbonic anhydrase is the book's example of one enzyme acting on related substrates.

Induced fit model: the flexible active site changes shape as the substrate binds, then returns to its original shape.

Comparison

Point	Lock and key model	Induced fit model
Proposed by	Emil Fischer (1894)	Koshland (1959)
Active site	Rigid; shape does not change	Flexible; moulded when the substrate binds
Fit	Complementary to the substrate from the start	Improves by a conformational change
Enzyme type	Non-regulatory (sucrase, maltase)	Regulatory or allosteric (hexokinase)

Exam focus

Explain the mechanism of enzyme action through the lock and key and induced fit models, with diagrams. (long)
A 7-mark Section C question in 2025 (Set D Q.4 OR); the book asks each model as a long question (long q4 and q5).
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Classify enzymes by the reaction they catalyse (six groups, one example each) and by the substrate they act on (four groups). (long)
Asked in 2026 (Set B Q.2 (ii), Set D Q.2 (ii)) and 2025 (Set B Q.2 (x), Set D MCQ 5); book long questions 13 and 14.
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Define minimum, optimum and maximum temperature and explain how temperature affects the rate of an enzyme reaction, with the graph. (short)
Asked in both 2026 sets (Set B Q.2 (vii) minimum, Set D Q.2 (vii) maximum) and book long question 8.
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Compare the types of cofactors (inorganic ions, coenzymes, prosthetic group) with an example of each; apoenzyme and holoenzyme. (short)
Asked in 2025 (Set B Q.2 (i) OR); book short questions 4 and 14 (b to d).
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Competitive inhibition: how a malonate-type inhibitor works, and how it differs from non-competitive inhibition. (mcq)
2025 Set B MCQ 8 (malonate and succinate dehydrogenase); book short question 14 (f) and long question 11.
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How an enzyme lowers the energy of activation (graph with the two humps). (mcq)
2026 MCQ 4 in both sets (Set B and Set D); book long question 7.
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Explain feedback inhibition with the threonine pathway, and non-competitive inhibitors such as cyanide and heavy metals. (long)
Not asked in 2025 or 2026 but a direct book long question 12 and short questions 12 and 13.
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Effect of pH, enzyme concentration and substrate concentration on the rate of enzyme action, with graphs. (long)
No paper question in 2025 or 2026; book long questions 9 and 10 and MCQ 4 and 14.
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