Enzymes I

Question 1

What are enzymes

Answer 1

Proteins that catalyse specific chemical reactions

Question 2

Example of enzyme functions

Answer 2

1. Digestion of biological molecules
2. Blood clotting
3. Defence-immune system
4. Movement: Actomyosin
5. Nerve conduction (e.g. Na+ pump)

Question 3

Examples of enzymes as drug targets

Answer 3

1. Antibiotics e.g. penicillin inhibit cell wall synthesis
2. Anti-inflammatory agents: Aspirin blocks prostaglandin synthesis
3. Anticancer drugs : methotrexate is a folate analogue: interferes with synthesis of DNA precursors

Question 4

State the key enzyme properties

Answer 4

1. Increase reaction rate by up to 10 billion fold
2. Show specificity
3. Unchanged at end of reaction
4. Do not alter reaction equilibrium
5. Facilitate reaction by decreasing the free energy of activation of the reaction

Question 5

What is an enzyme active site and how is it formed?

Answer 5

The active site is a 3D cavity or cleft that binds substrates with specificity through electrostatic, hydrophobic, hydrogen bonding and VdW interactions.

Question 6

How would you perform X-ray crystallography?

Answer 6

Crystallise your desired protein. Place the crystal in the path of the X-ray, and record the diffraction pattern. The spots in the pattern will have varying intensities and positions, use this information to identify the structure of the molecule.

Question 7

Describe the lock and key model

Answer 7

Enzyme active site directly complementary to substrate to form E-S complex

Question 8

Describe the induced fit model

Answer 8

Enzyme active site is not directly complementary, but as the substrate binds, the enzyme changes shape so the active site becomes complementary.

Example Hexokinase enzyme + Glucose

Question 9

In what ways do enzymes increase rate of reaction using E-S binding energy

Answer 9

1. To bring molecules together in the active site
2. To constrain substrate movement
3. To strain particular bonds in the substrate making breakage easier. Substrate is distorted on binding to resemble transition state
4. To stabilise positive and negative charges in the transition state
5. To exclude water from the active site- Make reaction go faster
6. To provide a reaction pathway of lower energy e.g. involving covalent enzyme-substrate intermediates
7. Use cofactors: Mg2+

Question 10

What is Vmax?

Answer 10

The maximum velocity of a reaction.

Question 11

What is Km?

Answer 11

The Michaelis Constant, which is half of the Vmax showing half the enzyme active sites are occupied. It measures the substrate binding affinity for the enzyme.

Question 12

How would you know an enzyme has a high substrate binding affinity?

Answer 12

Low Km

Question 13

What is the Michaelis-Menten equation?

Answer 13

V = Vmax * [S] / [S] + Km

Question 14

What is the Lineweaver-Burke equation?

Answer 14

1/V = 1/Vmax + Km/Vmax * 1/[S]

Question 15

What do the x and y intercepts of a Lineweaver-Burke double reciprocal graph give you?

Answer 15

Y intercept = 1/Vmax
X intercept = -1/Km

Question 16

What is kcat and how do we calculate it?

Answer 16

The kcat is the turnover number - the maximum number of substrate molecules handled per active site per second.

Vmax / [enz]

Question 17

What is competitive inhibition?

Answer 17

An inhibitor molecule competes with the substrate molecules for binding to the enzyme active site, forming an inactive EI complex and reducing rate of reaction.

Question 18

What is the effect of a competitive inhibitor on Km and Vmax

Answer 18

1. Km is increased (more substrate required to achieve Vmax/2)
2. Vmax is unaltered as the effects of the inhibitor can be competed out at high substrate concentrations.

Question 19

What is non-competitive inhibition?

Answer 19

The non-competitive inhibitor binds at the allosteric site of the enzyme, causing a change in shape so the active site can no longer bind with the substrate.

Question 20

How does non-competitive inhibition affect Km and Vmax

Answer 20

1. Km is unaltered
2. Vmax is reduced as the rate of reaction is reduced.

Question 21

What is allosteric regulation?

Answer 21

A regulatory molecule (acting at a pocket distinct from the active site) changes the enzyme conformation to influence the active site and decrease enzyme activity. Controls the flux of material through a metabolic pathway.

Question 22

How are metabolic pathways controlled?

Answer 22

Control achieved by allosteric regulation - Glycolysis has multiple control points including the early enzyme phosphofructokinase which is regulated by citrate, ADP and ATP.

Question 23

How else is enzyme activity regulated in cells

Answer 23

1. Control of gene expression (enzyme amount present in cells/secreted)
2. Compartmentation: Sequences in enzyme polypeptide chain target enzyme to ER, mitochondrion, nucleus etc
3. Covalent modification of enzyme. Change enzyme shape and activity- e.g. phosphorylation.