Enzymes

Question 1

Briefly describe the effect of an enzyme on a reaction

Answer 1

The activation energy is lowered in an enzyme-catalysed reaction. Thus, more reactant molecules can surmount the energy barrier to reach the transition state to be converted into product molecules

**The total energy difference/free energy change or Gibbs free energy between the reactant molecules and product molecules remains the same.

Question 2

Briefly describe the four categories of amino acid residues in an enzymes

Answer 2

1. Catalytic amino acid residues
   - The R groups of these amino acids are directly involved in the catalytic activity, ie. making or breaking of chemical bonds once substrate is bound.
2. Binding amino acids residues
   - The R groups of these amino acids hold the substrate(s) in position via non-covalent bonds while catalysis takes place.
3. Structural amino acid residues
   - Involved in maintaining the specific 3D conformation of the active site, as well as the enzyme as a whole.
4. Non-essential amino acid residues
   - Have no specific functions, and can be removed or replaced without the loss of the enzyme’s catalytic function.

Question 3

Name the three main types of cofactors:

Answer 3

Inorganic metal ions
Coenzymes
Prosthetic group

Question 4

Describe the characteristic and purpose of an inorganic metal ion cofactor in an enzyme

Answer 4

Mostly small divalent ions eg. Ca2+

May either be component of active site or affect enzyme activity through allosteric regulation.

Allosteric enzymes have multiple subunits and through conformational changes, bind activators of inhibitors at sites other than the active site. Aforementioned inhibitors usually bind reversibly to the enzyme and act by altering the enzyme’s active and/or allosteric sites to facilitate the catalytic reaction carried out by the enzyme.
Eg. salivary amylase activity is increased in the presence of chloride ions.

Question 5

Describe the characteristic and purpose of an coenzyme cofactor in an enzyme

Answer 5

Loosely associates with the enzyme during the reaction. Coenzymes act as transient carriers of specific functional groups, hydrogen or electrons. Most coenzymes are derived from vitamins.
Eg. Nicotinamide adenine dinucleotide (NAD) is an important coenzyme in respiration

Question 6

Describe the characteristic and purpose of an prosthetic group cofactor in an enzyme

Answer 6

Prosthetic groups are tightly bound to the enzyme on a permanent basis.
Eg. the prosthetic group of enzyme catalase is an iron-containing haem group

Question 7

What is a complete, catalytically active enzyme together with its bound coenzyme and/or metal ions called?

Answer 7

holoenzyme

Question 8

What is a holoenzyme?

Answer 8

A complete, catalytically active enzyme together with its bound coenzyme and/or metal ions

Question 9

What is an apoenzyme called?

Answer 9

It is the protein part of such an enzyme

Question 10

Name the classes of enzymes according to the types of reactions they catalyse (not impt?)

Answer 10

Oxidoreductase - Transfer of electrons (hydride ions or H atoms) aka oxidation-reduction reactions

Transferases - Transfer of functional groups

Hydrolases - Hydrolysis reactions (transfer of functional groups to water)

Lyases - Addition of groups to double bonds, or formation of double bonds by removal of groups

lsomerases - Transfer of groups within molecules to yield isomeric forms

Ligases - Formation of C-C, C-S, C-O, and C-N bonds by condensation reactions coupled to ATP cleavage

Question 11

Describe how enzymes lowers activation energy

Answer 11

Enzymes lower the activation energy. They do so by:

• orientating the substrates in close proximity, in the correct orientation, to undergo chemical reactions.
• straining critical bonds in the substrate molecule(s), allowing the substrates to attain their unstable transition state.
• providing a microenvironment that favours the reaction (eg. the presence of specific amino acids/ions at the active site may result in a specific set of molecular conditions that favours the formation I breakage of particular bonds).

Question 12

Explain enzyme specificity using the lock-and-key hypotheses

Answer 12

It suggested that there is an exact fit/complementary shape or conformation between the substrate and the active site of the enzyme, in the same way that a key fits into a lock very precisely.
The enzyme is viewed as a rigid structure, where only substrates that are exactly complementary to the conformation of the active site are able to bind to the active site for catalysis.
Thus, this explains substrate specificity of enzymes.

Question 13

Explain enzyme specificity using the induced-fir hypotheses

Answer 13

In the induced-fit hypotheses, the enzyme possesses active site flexibility.
The active site does not have a rigid conformation that fits only one type of substrate.
As such, it is rather flexible in conformation and can allow more than one type of substrate to bind.
Moreover, it is not in the precise complementary conformation to the substrate before binding to the substrate.

Upon binding of substrate, the active site changes its conformation slightly to bind the substrate even more firmly/snugly so that the R groups of the catalytic amino acids at the active site are:

• moulded into a specific conformation
• brought into close proximity to the chemical bonds in the substrate hence facilitating catalysis where the substrate is converted to product

Question 14

Explain the differences between the lock-and-key hypotheses and induced-fit hypotheses for enzyme specificity

Answer 14

The lock-and-key hypotheses explains substrate specificity while the induced fit hypothesis further explains group specificity. Where one enzyme is able to catalyse reactions for a variety of substrates that share similar structural or chemical properties.

In the lock-and-key hypotheses, the enzyme is viewed as a rigid structure, where only substrates that are exactly complementary to the conformation of the active site are able to bind to the active site for catalysis.

In the induced-fit hypotheses, there is active site flexibility, such that the active site does not have a rigid conformation that fits only one type of substrate. Instead, it is rather flexible in conformation and can allow more than one type of substrate to bind

Lastly, in the induced-fit hypotheses, it is not in precise complementary conformation to the substrate before binding to the substrate and changes its conformation slightly to bind to the substrate.

Question 15

Name the different methods of recording rate of an enzyme-catalysed reaction

Answer 15

Measuring rate of enzyme formation
Measuring initial rate of reaction
Measuring rate of substrate usage

Question 16

Briefly name all the factors affecting an enzyme reaction

Answer 16

Substrate concentration
Enzyme concentration
Temperature
pH

Question 17

Explain the effect on low substrate concentration on rate of enzyme-catalysed reaction

Answer 17

Increase in substrate concentration results in a proportional increase in the rate of reaction (substrate concentration is limiting factor).

Not all the active sites of the enzymes are occupied. Rate is limited by the concentration of substrate. An increase in substrate concentration increases the frequency of effective collisions between the enzyme active site and substrate molecules, hence increasing the number of enzyme-substrate complexes formed per unit time and consequently the amount of product formed per unit time, resulting in a proportional increase in the rate of reaction.

Question 18

Explain the effect of high substrate concentration on rate of enzyme-catalysed reaction

Answer 18

A point will be reached when any further increase in substrate concentration does not result in an increase in the rate of reaction (i.e. the graph reaches a plateau).

The active site of every enzyme molecule is occupied at any given moment. Rate is limited by saturation of enzyme active sites. ie. substrate availability is no longer the limiting factor.
Enzyme concentration is limiting. Any added substrates have to ‘wait’ until existing E-S complexes dissociate to release their products and free enzyme molecules in order to form new E-S complexes.
Further increase in substrate concentration does not result in an increase in the rate of reaction ie. graph reaches a plateau. Substrate concentration is no longer the limiting factor. Rate of reaction can only increase with the addition of enzyme.

Question 19

Explain the effect of low enzyme concentration on rate of enzyme-catalysed reaction

Answer 19

An increase in enzyme concentration results in a proportional increase in the rate of reaction as the concentration of enzyme is the limiting factor.

The increase in enzyme concentration provides more active sites, and therefore increasing the frequency of effective collisions between substrates and active sites. More enzyme-substrate complexes formed per unit time resulting in an increase in the amount of product formed per unit time. Hence, resulting in an increase in rate of reaction.

Question 20

Explain the effect of high enzyme concentration on rate of enzyme-catalysed reaction

Answer 20

A point will be reached when any further increase in enzyme concentration does not result in an increase in the rate of reaction ie. the graph reaches a plateau.

Enzyme concentration is no longer a limiting factor. There are not enough substrate molecules competing for the active sites available. Substrate concentration is limiting. Rate of reaction can be increased with the addition of substrate

Question 21

Explain the effect of temperature increasing on rate of enzyme-catalysed reaction

Answer 21

As the temperature increases to the optimum temperature from 5°C to 40°C, The rate of reaction increases.
At low temperatures near or below freezing point, enzymes are inactivated. Increasing temperature increase the kinetic energy of the substrate and enzyme molecules, thereby increasing the frequency of effective collisions between substrates and active sites, increasing the formation of enzyme-substrate complexes per unit time and the amount of product formed per unit time. This increases the rate of reaction.
Enzyme activity is highest at its optimum temperature of 40°C for most cases. The rate of ES complexes formation is the highest at optimum temperature

If the temperature is increased beyond the optimum temperature of 40°C, a decrease in the rate of reaction occurs despite the increasing frequency of collisions.
Thermal agitation of enzyme molecule disrupts the hydrogen bonds, ionic bonds and other non-covalent interactions that stabilise the specific 3D conformation of the protein molecule. This results in the loss of the 3D conformation of the enzyme and that of its active site so that there is no longer a complementary fit with the substrate. The enzyme is said to be denatured and loses its catalytic function.
The frequency of effective collisions between substrates and active sites decrease and the rate of formation of enzyme-substrate complexes drops. Hence, less product is formed per unit time.

Question 22

Explain the effect of deviation in pHon rate of enzyme-catalysed reaction

Answer 22

Changes in pH can affect enzyme activity by altering the ionic charge of the acidic and basic R groups of proteins:
- Lower pH&raquo_space; more H+ ions available to neutralise negative charges present in the enzyme.
- Higher pH&raquo_space; less H+ ions available to neutralise negative charges present in the enzyme.
Change in ionisation of amino acids disrupts the ionic bonds/hydrogen bonds that maintain the 3D conformation of the enzyme, denaturing the enzyme.

Question 23

Briefly explain the rationale behind an optimum pH

Answer 23

At the optimum pH, the rate of reaction is at a maximum .
Intra-molecular bonds are intact and conformation of active site is ideal for binding, therefore frequency of effective collision is the highest with largest amount of enzyme-substrate complexes formed.

Question 24

What is the Michaelis Constant?

Answer 24

Km is measured as the substrate concentration that allows an enzyme-catalysed reaction to proceed at half the maximum velocity, 1/2Vmax

Question 25

Describe the structure of competitive inhibitors with reference to the binding sites of the inhibitor

Answer 25

Competitive inhibitors are structurally similar to the substrate molecule and compete with the substrate for binding to the active site. Although it is not acted upon by the enzyme, it remains bound to the active site and prevents substrate binding to active site.

Question 26

Describe the structure of non-competitive inhibitor with reference to the binding sites of the inhibitor

Answer 26

Non-competitive inhibitors bear no structural resemblance to the substrate. It does not compete with the substrate for the active site. However, it binds to a part of the enzyme molecule that is not the active site which alters the 3D conformation of the enzyme molecule and active site.
The enzyme molecule no longer has an active site that is complementary in conformation to the substrate. Hence substrate does not bind to the enzyme active site and no enzyme-substrate complex can be formed

Question 27

Explain the effects of competitive inhibitors on the rate of enzyme activity

Answer 27

The initial rate of reaction is reduced in the presence of competitive inhibitor. However, when [S] is very high, both reactions reach the same Vmax. However in the presence of the inhibitor, this requires a longer period of time to
produce the same amount of product.

An increase in substrate concentration reduces the effect of inhibition. This is because the substrate and the inhibitor are in direct competition for the enzymes’ active sites and the greater the proportion of the substrate molecules, the greater the chance a substrate can out-compete the inhibitor to enter the active site. If so, a rate of reaction almost equivalent to Vmax can be attained. The final amount of product formed is the same as the substrate continues to be converted by any enzymes molecules that are unaffected by the inhibitor .

**[S] - Concentration of substrate
Vmax - Maximum velocity of RoR

Question 28

Explain the effects of non-competitive inhibitors on the rate of enzyme activity

Answer 28

The initial rate of reaction is reduced in the presence of non-competitive inhibitor. Even when [S] is very high, the initial rate of reaction in the presence of inhibitor does not reach the same Vmax as the reaction that is uninhibited.

The binding of non-competitive inhibitor to a site other than the enzyme’s active site causes a change in 30 conformation of the enzyme’s active site. Hence, prevents substrate molecules from binding. A certain proportion of the enzyme molecules are rendered inactive, Vmax is lower. As the substrate and the inhibitor are not in direct competition for the same site, an increase in substrate concentration has no effect on the inhibition. Note that Km remains unchanged, as the affinity of the enzyme for substrate remains unaffected. The final amount of product formed is the same as the substrate continues to be converted by any enzymes molecules that are unaffected by the inhibitor.

**[S] - Concentration of substrate
Vmax - Maximum velocity of RoR
Km - Michaelis Constant

Question 29

Explain the effects of allosteric inhibitors/activators on the rate of enzyme activity

Answer 29

Allosteric activation
When an activator binds, it stabilizes the active form of the enzyme and increases the affinity of the enzyme for its substrate

Allosteric inhibition
When an inhibitor binds to the same region, it stabilises the inactive form of the enzyme and decreases the affinity of the enzyme for its substrate

The graph for an allosterically activated enzyme reaction is sigmoidal in shape. The binding of an activator to an allosteric site induces a favourable conformation change in the active sites of all the subunits of the enzyme. This significantly amplifies the response of the enzyme to substrates. The amplification results in the sudden steep rise in the rate of reaction.

Question 30

Explain the theory of cooperativity in enzymes

Answer 30

Binding of one substrate molecule to an active site of a multimeric enzyme triggers the same
favourable conformation change in the active sites of all other subunits of the enzyme.

Likewise, cooperativity amplifies the response of enzyme to substrate. One substrate molecule primes an enzyme to accept additional substrate molecules .

Question 31

Name the differences between reversible and irreversible inhibition

Answer 31

The reversible inhibitors binds to the enzyme via weak non-covalent bonds such hydrogen bonds, hydrophobic interactions, While irreversible inhibitors bind to the enzyme via covalent bonds.

The effect of reversible inhibitors is temporary, the inhibitor can be easily removed and cause no permanent damage to the enzyme, such that when the inhibitor leaves, activity of the enzyme will be restored to normal. Irreversible inhibition causes permanent damage to the enzyme molecule so that it is unable to carry out catalytic activity.