Enzymes 1

Question 1

Define and describe enzymes

Answer 1

Enzymes are proteins catalysts, which increase the rate of reactions without being changed in the overall process

• they increase the rate of chemical reactions between substrates (reactants) and products
• can be modified during the reaction, but always return to their original form

They are key players in metabolic pathways

Question 2

Describe substrate-binding sites as a key concept of enzymes (active sites)

Answer 2

Substrates bind to specific sites through interactions with amino acids, coenzymes and metal ions
- the spatial geometry (3D structure) dictates specificity

Question 3

Describe activation energy and transition states, in relation to enzymes

Answer 3

The binding of substrates promotes the formation of a transition state which lower activation energy

Question 4

Describe pH and temperature profiles

Answer 4

Both pH and temperature affect enzyme activity
- Reaction rate increases with temperature
BUT
enzyme stability decreases (denaturation)

Question 5

Name 2 factors affecting reaction velocity/rate of reactions (in relation to enzymes)

Answer 5

Temperature

pH

Question 6

Describe how the velocity of reaction can increase with temperature

Answer 6

Reaction velocity increases with temperature

• until a peak velocity is reached
• due to the increased number of substrate molecules having sufficient energy to pass over the energy barrier + form products of the reaction

Question 7

Describe how the velocity of reaction decreases with higher temperature

Answer 7

Further elevation of the temperature causes a decrease in reaction velocity
- due to temperature-induced denaturation of the enzyme

Question 8

Describe how pH affects the reaction rate (in relation to enzymes)

Answer 8

The concentration of protons affects amino acids in the enzyme, by altering the interactions between charged R groups in the polypeptide, altering the shape of the enzyme
- leading to denaturation of the enzyme

Question 9

Describe the enzyme catalytic cycle

Answer 9

3 Basic steps:

1. the substrate binds
2. product forms
3. Product unbinds

Enzymes do not alter the position of the reaction equilibrium, they just accelerate the establishment of this equilibrium

Question 10

Name the two substrate-binding models

Answer 10

• The ‘Lock and Key’ model (old)

- The ‘induced-fit model (current)

Question 11

Describe the Lock and Key model of substrate binding

Answer 11

A complimentary 3D surface recognises the substrate

• substrate binds through Hydrogen bonds and hydrophobic/hydrophilic interactions
• Binding can be prevented by steric hindrance and charge repulsions

Question 12

Describe the ‘induced-fit’ model of substrate binding

Answer 12

As substrates bind, enzymes undergo a conformational change

• side chains of (active sites) amino acids reposition
• binding interactions increase
• not a rigid lock - but a dynamic surface

Question 13

Name the different energy changes that occur during the reaction

Answer 13

1. Activation energy
2. Rate of reaction
3. Alternate reaction pathway

Question 14

Define transiton state

Answer 14

The transition state is the point of maximal bond strain

Question 15

Describe activation energy

( in relation to energy changes during a reaction)

Answer 15

Activation energy

• Difference in free energy between the reactant (substrate) and transition state
• because of high Ea, the rates of uncatalysed chemical reactions are often slow

Question 16

Describe the rate of reaction (in relation to energy changes during a reaction)

Answer 16

For molecules to react, they must contain sufficient energy to overcome the energy barrier of the transition state
- Without an enzyme, only a small proportion of molecules possess enough energy to achieve the transition state between reactant and product - the rate of reaction is determined by the number of such molecules

So, the lower the Ea, the more molecules have sufficient energy to pass the transition state, so the faster the rate of reaction

Question 17

Describe the alternative reaction pathway an enzyme provides

Answer 17

An enzyme allows a reaction to proceed rapidly under conditions prevailing in the cell by providing an alternate reaction pathway with a lower Ea

Question 18

Describe cofactors and coenzymes

Answer 18

Catalytic properties are often dependent on non-peptide molecules called cofactors

• Metal ions and organic coenzymes
• e.g. NAD+, NADP+, Mg2+

Tightly bound cofactors are known as ‘prosthetic groups’
- e.g. FMN, FAD, Fe2+, ZN2+

In humans, coenzymes are usually synthesised from vitamins (so dietary deficiencies can cause problems)

Coenzymes have little to no activity and specificity on their own. Binding to enzymes provides orientation and stabilisation

Question 19

Describe how a cofactor/coenzyme is involved in activation/transfer

Answer 19

They participate directly in catalysis by forming a covalent bond between the coenzyme’s functional group and the substrate (a separate portion binds to the enzyme)

Question 20

Describe how cofactors/coenzyme in Oxidation-Reduction reactions

Answer 20

e.g. NAD+ and FAD+

The cofactor is involved in either the oxidation (loss of electrons) or reduction (gain of electrons) of the substrate

Question 21

Describe how metal ion cofactors help in enzyme activity

Answer 21

Positively charged metal ions act as electrophiles (electron-attracting groups

e. g. Alcohol Dehydrogenase utilises zinc to transfer electrons from ethanol to NAD+
- it forms acetaldehyde and NADH
- Active site contains ‘activated’ serine
- This -ve charge is stabilised by Zn2+

Question 22

Define isoenzymes

Answer 22

Isoenzymes

• Enzymes that differ in sequences (i.e. different genes) but catalyse the same reaction
• They can have different kinetic parameters (Km and Vmax) and substrate specificities

Question 23

Describe multi-enzyme complexes

Answer 23

Some enzymes catalysing multiple consecutive steps in metabolic pathways associate to form multi-enzyme complexed

Advantages:

• reduced transit time between steps
• less potential for interference (products acted on by correct enzyme)

Question 24

Describe diagnostic enzymology

Answer 24

It is the measurement of enzyme activity or concentrations in serum

• can include normal serum enzymes (e.g. clotting factors), secreted enzymes (e.g. pancreatic lipase) and enzymes released by damaged or malignant cells
• Cell leakage can be caused by reduced O2 (ischaemia, impairs energy production), toxic chemicals, microorganisms, immune responses and genetic conditions

Released enzymes are affected by metabolism and excretion, they, therefore, have different serum half-lives:

• intestinal alkaline phosphatase (few hours)
• liver alkaline phosphatase (few days)

Question 25

Describe the use of diagnostic enzymology in ischaemic heart disease

Answer 25

Heart cells depend on oxygen, which can be restricted by cholesterol-rich atheromatous plaques causing:

• stable angina (angina pactoris)
• myocardial infarction (causing irreversible cardiac cell damage)

Cell contents are released over hours, including creatine kinase (CK) and troponin T or I which can be monitored

Creatine kinase (CK) is a dimer of M (muscle) and B (Brain) subunits

• heart muscle - MM (85%) and MD (15%)
• skeletal muscle - MM (99%)
• Brain, stomach, intestine, bladder mainly BB

As CK-MB is heart-specific, raised serum levels are symptomatic of myocardial infarction

• an increase in total creatine kinase activity can be detected within 6 hours of a heart attack
• CK serum levels peak at 24-36 hours
• CK-MB is detectable within 3-4 hours, with a peak at 100-24 hours
• remains elevated for 2-4 days

Question 26

Describe enzyme kinetics

Answer 26

E + S [ES] E + P

• Like all chemical reactions, catalysed reactions move towards the equilibrium, where substrates and products are in a steady-state
• The most common approach to analysing enzyme kinetics is using initial rate, v0
• this is affected by [S] and [enzyme], along with external factors (temperature etc.)

Question 27

Describe how the initial rate is related to Vmax and Km

Answer 27

Plotting v0 against [S] with a fixed [enzyme] yields a hyperbolic curve, which shows how reaction velocity varies with [substrate]

Michaelis-Menten equation

v0 = (Vmax[S])/(Km + [S])

The equation relates initial velocity to [substrate and 2 other parameters]:

• Vmax is the maximum possible rate at that [enzyme]
• Km is the [substrate] resulting in Vmax/2

Question 28

Define and describe Vmax

Answer 28

It is the theoretical rate of an infinitely high substrate concentration
- where all the enzyme molecules contain a bound substrate

The Vmax is directly proportional to the amount of enzyme used

Question 29

Describe Km

Answer 29

Km (Michaelis constant)

• Defined as the substrate concentration at which initial rate is 1/2 of Vmax
• enzyme velocity is most sensitive to changes in [Substrate] close to Km
• If Km is known, the [substrate] required to saturate the enzyme can be calculated

High Km = low substrate efficacy

Question 30

Give assumptions that are being made in order for the Michalis Menten equation to work

Answer 30

• A single substrate reaction where the enzyme-substrate complex (ES) can freely dissociate
• [Substrate]&raquo_space; [Enzyme]
  therefore [S]&raquo_space; [ES]
• not applicable to all enzymes (e.g. multi-enzyme complexes)