Enzyme Kinetics & Inhibitors

Question 1

Why is the rate of biological reactions important?

Answer 1

Most cellular chemicals are unstable but rely on the rate of their breakdown being too slow to matter (without catalysis).
-some diseases are caused by imbalances in the rates at which reactions are taking place.

Question 2

How can you speed up a reaction?

Answer 2

• increase temp
• increase pressure
• change pH
• BUT cells have to work at pH 7.0, at ~310K, and at room pressure.
• therefore you must lower the transition state energy.

Question 3

How do enzymes increase the rate of reactions?

Answer 3

reduce the transition state energy for a reaction.

Question 4

Give the general properties of enzymes.

Answer 4

• catalytic = ends in the state that it started
• specific = changes the rate of a limited set of reactions
• fast = most reactions need to be speeded up by many orders of magnitude

Question 5

Why proteins as enzymes?

Answer 5

• proteins are good at accommodating different reactants so that the protein itself is not changed in the reaction (catalytic).
• 20 amino acids offers a wide possibility of differences in shape (specific).
• the amino acids have very different chemical properties so more reactions can be catalysed (speed).

Question 6

How is the transition state barrier reduced?

Answer 6

-stabilise the unfavourable intermediate
Charge-charge interactions
H bonding
Protecting hydrophobic groups
-make a possible less favourable reaction
Provide acid/base like conditions
Allow oxidation/reduction
Provide a small vacuum
Provide an attacking group (covalent catalysis)
Provide a metal ion

Question 7

For enzyme kinetics what can we assume?

Answer 7

• release of the product is very fast (compared to the reaction itself).
• the reverse reaction is sufficiently slow that we can ignore it, or the product is reacting with something else so that it does not remain.

Question 8

Define Km

Answer 8

• values of enzymes range widely.
• for most enzymes, lies between 10-1 and 10-7 M.
• depends on the substrate and conditions such as pH.
• is the concentration of substrate at which ½ the active sites are filled.
• provides a measure of the substrate concentration required for significant catalysis to occur.

Question 9

Km is also a measure of…

Answer 9

the strength of the ES complex.

indicates the affinity of the ES complex only when K1 is much greater than K2

Question 10

A high Km value indicates _ binding

Answer 10

weak binding

Question 11

Vmax reveals…

Answer 11

the turnover number, Kcat.

Question 12

The kinetics of an enzyme will differ if…

Answer 12

• the enzyme is affected by the concentration of some other compound.
• the reaction is more complicated than the simple scheme shown.
• the enzyme has multiple subunits.
• the laws of mass action do not apply.
• many other situations can demand modifications to these kinetics.

Question 13

Kcat/Km is a measure of…

Answer 13

catalytic efficiency.

Question 14

Define an inhibitor.

Answer 14

• any molecule that acts to reduce the rate of an enzymatic reaction.
• act in a number of different ways to achieve this effect.
• can be small chemicals or larger polymers (inc other proteins).

Question 15

Define competitive inhibitors & give an example.

Answer 15

• usually resembles the substrate shape so that it specifically binds to the active site but also differs from the substrate so that it cannot react as the substrate does.
• many drugs act as competitive inhibitors
• active sites are easy to target, as the substrate is known & so related molecules can be designed.
• a well-designed inhibitor can greatly increase the KM of the enzyme
• eg Viagra (sildenafil) which mimics the substrate of phosphodiesterase

Question 16

How does irreversible inactivation resemble non-competitive inhibition?

Answer 16

-if an inhibitor binds irreversibly to an enzyme, it is classed as an inactivator.
-inactivators truly reduce the effective level of [E]T and therefore Vmax, at all values of [S] without changing KM.
the double reciprocal plots for irreversible inactivation are similar to those of non-competitive inhibition (the lines intersect on the 1/[S] axis.)
-reagents that chemically modify specific amino acid residues can act as inactivators.
-eg compounds (DIPF) used to identify the catalytic Ser and His residues of serine proteases are inactivators.

Question 17

How does competitive inhibition affect Km?

Answer 17

increases

Question 18

How does competitive inhibition affect Vmax?

Answer 18

none

Question 19

How does uncompetitive inhibition affect Km?

Answer 19

decreases

Question 20

How does uncompetitive inhibition affect Vmax?

Answer 20

decreases

Question 21

How does mixed inhibition affect Km?

Answer 21

increases

Question 22

How does mixed inhibition affect Vmax?

Answer 22

decreases

Question 23

How does non-competitive inhibition affect Km?

Answer 23

none

Question 24

How does non-competitive inhibition affect Vmax?

Answer 24

decreases

Question 25

Define multi-subunit enzymes & give an example.

Answer 25

-many enzymes are made from more than one protein chain (quaternary structure).
-these chains can be identical or different.
-in some cases, the binding of substrate to one subunit influences the binding to other subunits.
-results in cooperativity between the subunits.
Eg aspartate transcarboxylase

Question 26

How can enzyme activity be regulated.

Answer 26

-control of enzyme availability
The amount of enzyme in a cell depends on both its rate of synthesis & degradation. These rates are controlled by the cell & are subject to dramatic changes over time.
-control of enzyme activity
Can be directly regulated through structural alterations that influence the enzymes substrate binding affinity. Allosteric mechanisms can cause large changes in enzymatic activity.

Question 27

Define active sites.

Answer 27

• in most enzymes, the binding of the substrate & catalysis are carried out by around 20-30 amino acids.
• average enzyme size is 300 amino acids.
• active site = region where biding of substrate & catalysis take place.
• other parts of the protein are present to provide the protein with its overall structure, to ensure the active site has correct conformation & allow for regulation.

Question 28

Define allosteric effectors.

Answer 28

• any molecule that can bind to an enzyme away from the active site, and by doing so change the enzymes activity for its substrate.
• they can either increase the rate of reaction (allosteric activator), or reduce it (allosteric inhibitor).

Question 29

How can allosteric effectors work?

Answer 29

• proteins have inherent flexibility because of their small groups of secondary structure connected by loops.
• small changes in the structure in one part of the protein can be communicated throughout the entire structure.
• these changes can affect the fit of the active site to the substrate & the transition state.

Question 30

Define allostery.

Answer 30

-situation where a chemical binds to an enzyme away from the active site & this influences the kinetic properties of the enzyme.