6 - Mechanism of enzyme catalysis

Question 1

What is a catalyst and what does it do?

Answer 1

Catalysts stabilise the transition state of a specific reaction.

A catalyst:

• Lowers activation energy increasing ROR.

Is not consumed during the reaction.

Does not affect the eq. position.

Question 2

What is a protein?

Answer 2

A protein is a large organic compound made of amino acids arranged in a linear chain and folded into a 3D structure

Question 3

How does an active site form on a protein?

Answer 3

Protein folding brings together side chains of various amino acids that may be far apart in the primary sequence into close juxtaposition forming an active site.

Question 4

What are the 4 stages of an enzyme-catalysed reaction?

Answer 4

1. Enzyme + substrate
2. Enzyme-substrate (E-S complex)
3. Enzyme-product (E-P complex)
4. Enzyme + product.

Question 5

What are the 5 properties of the active site?

Answer 5

1. Positions the substrate molecules in the most favourable orientation for reaction
2. Perfectly complementary to transition state.
3. Amino acid side chains of the AS stabilise the electron distribution of the TS.
4. The substrate is strained on binding with AS lowering Ea and increasing ROR.
5. The products bind less tightly to the AS than the substrates so are released.

Question 6

What non-covalent interactions occur between the substrate and the amino acid side chains of the enzyme?

Answer 6

ionic bonds:
- Acidic groups (Asp, Glu)
- Basic groups (Lys, His, Arg)

hydrophilic interactions:
- OH groups (Ser, Thr, Tyr) - SH groups (Cys) -
- Amide groups (Asn, Gln)

Hydrophobic interactions:
- Ala, Leu, Leu, Ile, Val, Met

Aromatic interactions:
- Aromatic groups (Phe, Try, Trp)

Question 7

What are 3 ways in which reactive groups at the catalytic site surface catalyse a reaction?

Answer 7

• Donating or withdrawing electrons
• Stabilising or generating free radical intermediates
• Forming temporary covalent bonds

Question 8

What are cofactors, 3 examples of what they can be and 3 specific examples?

Answer 8

• Metal group such as (Mg2+)
• Prosthetic group - covalently bound organic molecule (haem)
• Coenzyme - tightly but not covalently bound organic molecule (NAD)

Question 9

What are enzymes called when they are catalytically active and when they aren’t?

Answer 9

Enzyme with prosthetic group/ coenzyme - holoenzyme (active)

Enzyme without prosthetic group / coenzyme - apoenzyme (inactive)

Question 10

What reactions do oxidoreductases catalyse?

Answer 10

Oxidation and reduction

Question 11

What reactions do transferases catalyse?

Answer 11

Transfer a chemical group from one substrate to another.

Question 12

What reactions do hydrolases catalyse?

Answer 12

Hydrolysis reactions

Question 13

What reactions do lysases catalyse?

Answer 13

Addition across a C=C bond

Question 14

What reactions do isomerases catalyse?

Answer 14

Intramolecular rearrangements

Question 15

What reactions do synthetases catalyse?

Answer 15

Formation of bonds between two substrates

Question 16

What is enzyme activity and what is it measured in ?

Answer 16

Number of micromoles (µmol) of substrate converted per minute under standard optimised conditions at 30°C.

Enzyme unit (EU) = µmol min-1

Question 17

What is specific activity and what is it measured in?

Answer 17

Measures enzyme purity - activity of enzyme per mg of total protein in the enzyme preparation.

measured in - µmol min-1 mg-1

Question 18

What 3 factors must be fulfilled for a meaningful quantitative assay of enzyme activity?

Answer 18

• Measured at fixed [enzyme]
• Measured at defined temperature and pH
• initial velocity V0 is measured.

Question 19

What 5 factors affect enzyme activity?

Answer 19

• pH
• Temperature
• [Enzyme]
• [Substrate]
• Covalent modification of enzyme

Question 20

Why does pH affect enzyme-catalysed reactions?

Answer 20

pH effects the ionisation state of amino acid side chain affecting the binding of the substrate.

Question 21

What is enzyme denaturation?

Answer 21

Denaturation can occur at high temperatures and at extreme pH levels.

The extreme conditions lead to a loss of hydrogen bonding causing the enzyme to unfold resulting in a loss of activity.

Question 22

Why does temperature affect enzyme-catalysed reactions?

Answer 22

Higher temperatures increase ROR
- Molecules move faster, greater collision chances, electrons gain Ea easier.

Temperature optimum depends on the time of incubation.

Question 23

What effect does varying enzyme concentration have on enzyme-catalysed reactions?

Answer 23

Typically increasing enzyme concentration causes a linear increase in product formation.

In some situations the enzyme can associate or dissociate into a different form (monomer or dimer) which may be more or less active at different enzyme concentrations.

This can cause the linear increase to become curved - either increasing faster or dropping off at higher concentrations.

Question 24

What effect does varying substrate concentration have on enzyme-catalysed reactions?

Answer 24

An increase in substrate concentration increases the rate of reaction until the enzyme becomes saturated at which point the ROR begins to level.

Question 25

How is the Michaelis constant determined and what does a high and low Km suggest?

Answer 25

The Michaelis constant (Km) is the concentration of substrate required to achieve half (V1/2) the maximum rate of reaction (Vmax).

High Km - low substrate affinity. (High ROR volatility due to low [S] change)

Low Km - High substrate affinity. (little to no ROR volatility due to moderate [S] change)

Question 26

What is the Michaelis-Menten equation and what does it measure?

Answer 26

Describes the dependence of ROR on concentration of substrate

v (rate of reaction) = Vmax x [S] / Km + [S]

Question 27

How can the Km and Vmax of an enzyme be determined experimentally?

Answer 27

The initial rate of reaction of the enzyme under optimal conditions should be determined.

Reaction rate should be measured at a range of substrate concentrations.

The lineweaver-Burk double reciprocal plot of reaction rate over substrate concentration can be used to determine Km and Vmax.

Question 28

What are the 2 different ways in which an enzyme with two substrates can react when both are present?

Answer 28

1. Sequential reaction - each substrate binds in turn.
2. Ping-pong reaction - one substrate reacts and modifies enzyme, then substrate reacts with modified enzyme.

Question 29

What are allosteric enzymes?

Answer 29

Allosteric enzymes contain other than substrate binding sites.

often in multi-subunit complex with multiple active sites.

Binding of substrate to the active site of one subunit leads to change in conformation activating other active sites in the complex.

Question 30

how do allosteric enzymes differ from Michaelis-Menten enzymes when on a ROR-[S] graph?

Answer 30

On a ROR-[S] graph the shape of the curve hyperbolic for M-M enzymes and is sigmoidal for allosteric enzymes.

Question 31

What are the two types of enzyme inhibitor and 3 characteristics of each?

Answer 31

Reversible inhibitors
- Bind to enzyme non-covalently
- Relatively unspecific
- Blocks substrate binding

Irreversible inhibitors
- Bind to enzyme covalently
- Substrate analogues
- Transition state intermediate does not break down

Question 32

How do competitive inhibitors work?

Answer 32

They compete with the substrate for binding at the active site.

Question 33

How do mixed inhibitors work?

Answer 33

Mixed inhibitors do not bind in the active site.

Mixed inhibitors distort the substrate binding site affecting substrate affinity and catalytic turn-over.

Question 34

How do non-competitive inhibitors work?

Answer 34

BInds to the enzyme at a position separate from the active site.

The affinity for the substrate is unchanged but the rate of reaction is slowed.

Question 35

What effect do competitive, non-competitive and mixed inhibitors have on Km and Vmax

Answer 35

Competitive - Vmax: unchanged Km: Increased

Non-competitive - Vmax: decreases Km: unchanged

Mixed - Vmax: decreases Km: Increase or decrease.