CBS - Enzyme Properties/Kinetics/Regulation

Question 1

Define (and describe) enzymes.

Answer 1

Enzymes are biological catalysts which speed up the rate of a reaction, without altering the final equilibrium between reactants and products.

They are regenerated at the end of the reaction.
They are typically proteins (though not only – ribozymes are involved in RNA splicing, and are RNA based).

Question 2

List some key attributes of enzymes.

Answer 2

• speed
• selectivity
• specificity

Question 3

What determines enzyme substrate specificity?

Answer 3

Specificity is determined by the presence of a groove or cleft of defined shape called the ‘active site’ into which only the substrate of the correct shape and charge can fit.

Question 4

List the 6 classes of enzymes, what they do, and an example of each.

Answer 4

1. Oxidoreductases: catalyze the transfer of hydrogen atoms and electrons (e.g. lactate dehydrogenase)
2. Transferases: catalyze the transfer of functional groups from donors to acceptors (e.g. alanine amino transferase (ALT) )
3. Hydrolases: catalyze the cleavage of bonds by the addition of water (hydrolysis) (e.g. trypsin)
4. Lysases: catalyze the cleavage of C-C, C-O, or C-N bonds (addition of groups to double bonds or formation of double bonds by removal of groups) (e.g.ATP-citrate lyase)
5. Isomerases: catalyze the transfer of functional groups within the same molecule (e.g. phosphoglucose isomerase)
6. Ligases: use ATP to catalyze the formation of new covalent bonds (e.g. DNA ligase)

Question 5

Describe the refined key and lock theory - the ‘induced fit’ theory.

Answer 5

Enzymes will undergo conformational changes upon substrate binding, induced by weak interactions with the substrate itself

These changes can affect residues in the active site as well as repositioning entire domains.
The “induced fit” serves to bring specific
functional group within the enzyme in the
proper position to catalyse the reaction

The new enzyme conformation has
enhanced catalytic properties.

Question 6

What is chymotrypsin?

Answer 6

Chymotrypsin is a digestive enzyme which breaks down proteins in the small intestine. It is secreted by the pancreas and converted into an active form by trypsin.

Question 7

Describe the catalysis of the peptide by chymotrypsin (in detail).

Answer 7

1. The key residues that allow catalysis to take place is the catalytic triade. It is made up of serine, histidine and aspartate.

The hexagon is the element of specificity that allows the substrate to be recognised by chymotrypsin. The residue will fit into a hydrophobic pocket that stabilises the sequence, thereby provided the bond to the catalytic triade.

Thus, when the substrate binds to the enzyme, we have the formation of the enzyme-substrate complex.

2. Once that is formed, we have an attack by the nucleophile to the C alpha atom of the substrate. This is allowed by the interaction of the nucleophile to the histidine, which is stabilised by the aspartate. Aspartate stabilises the formation of the positively charged histidine that has taken the proton from the nucleophile, so that it can attack the C alpha of the peptide, forming the tetrahedral intermediate.
3. Once the proton is transferred to the C terminal fragment, then we have the formation of the acyl peptide intermediate, which can go and leave the active site.

The acyl intermediate has allowed the formation of this free peptide which can leave.

4. The free peptide is replaced by a water molecule, which is hydrogen bonded to the histidine.
5. Similar to before, the histidine protonated the water, which can preform a nucleophilic attack on the C alpha, giving rise to a second tetrahedral intermediate.
6. Now, this allows the portion to leave the active site, and we have regenerated the enzyme for the beginning of another reaction.

Question 8

List some different ways to plot enzyme rate.

Answer 8

• Michealis-Menten plot (most commonly used - shows hyperbolic behaviour)
• Linewaver-Burk (double reciprocal) plot - a very clear way of identifying different inhibition, but replies on extrapolation and can be influenced by poor data distribution
• Eadie-Hofstee plot (v/[S] against v)
• Hanes plot
  ([S]/v against [S])

Question 9

What does Vmax represent?

Answer 9

Vmax is the highest rate, which is when the entire enzyme-substrate complex is equal to the total concentration of enzyme.

If we saturate the total concentration of enzymes with substrate, then it is not possible for the reaction to go faster than that, so it is considered Vmax.

Question 10

What does Km represent?

Answer 10

Km corresponds to the substrate concentration at the point at which the initial velocity corresponds to Vmax/2.

Under steady state conditions Km is a measure of the lifetime of the ES complex and gives an indication of the substrate concentration required for significant catalysis.

We can loosely say that the lower the value, the higher the affinity for an enzyme to a particular substrate.

Question 11

What does Kcat represent?

Answer 11

Kcat gives us the number of molecules of substrate against turnover into product as a function of unit of time.

Question 12

What does Ka represent?

Answer 12

This is the specificity constant or catalytic efficiency and is useful for examining enzyme kinetics when [S] &laquo_space;Km (it is also the second-order rate constant for the reaction at low substrate concentration).

The higher the Ka value, the better the enzyme.

Question 13

List the different ways to regulate enzymatic activity.

Answer 13

Environmental:

• location
• time
• temperature
• pH

Enzyme inhibition:

• reversible - irreversible
• competitive - noncompetitive - uncompetitive

Allosteric binding sites:
- postives and negative effectors at different sites
- multiple subunits
cooperative kinetics

Question 14

Define reversible inhibitors.

Answer 14

Reversible inhibitors bind to the enzyme molecule but can dissociate again.

• when they bind, the enzyme has low or no activity
• when they dissociate the enzyme activity is restored

Question 15

Define irreversible inhibitors.

Answer 15

Irreversible inhibitors bind to the enzyme, which is permanently inactivated as a result. Often the binding is slow, so the activity of the enzyme decays with
time.

Question 16

Give an example of an irreversible inhibitor.

Answer 16

An example of an irreversible inhibitor is when di-isopropyl phosphofluoridate binds to chymotrypsin and chemically modifies an active-site residue (serine)

For the inhibition of serine, the fluoride gets released and this forms a phosphoester bond with the serine, and the covalent bond becomes so strong it cannot be removed, so it stays there blocking the active site and blocking enzyme activity.

Question 17

List and define the different types of inhibition.

Answer 17

COMPETITIVE - They are inhibitors that bind directly to the active site of an enzyme. The competitive inhibitor competes with the substrate to bind to the enzyme. A competitive inhibitor typically mimics the substrate, competing for the active site.

NON-COMPETITIVE - A non-competitive inhibitor binds to the enzyme away from the active site, altering the shape of the enzyme so that even if the substrate can bind, the active site functions less effectively.

UNCOMPETITIVE - An uncompetitive inhibitor is an inhibitor that only binds to the enzyme-substrate complex. The formation of its binding site only forms when the enzyme and the substrate have interacted amongst themselves. The uncompetitive inhibition does not work when additional substrates are trying to be involved. The enzyme-substrate-inhibitor complex does not produce any product.

Question 18

Give an example of competitive inhibition.

Answer 18

succinate dehydrogenase is an enzyme that works via competitive inhibition.

It catalyses the oxidation of succinate to fumarate, and is inhibited reversibly by malonate. This is because it resembles the substrate and can’t be oxidized.

Question 19

What effect does competitive inhibition have on Km and Vmax?

Answer 19

It increases Km but does not affect Vmax.

As we increase the concentration of the inhibitor, Km becomes progressively higher, meaning that there is a lower apparent affinity for the substrate to the active site.

However, since there is always a chance that the inhibitor can be displaced out and replaced with proper substrate, the Vmax is unaltered.

Question 20

Describe the fluoride inhibition of enolase.

Answer 20

Enolase is a key enzyme of glycolysis.

It catalyses the formation of phosphoenolpyruvate (PEP).

F- is a non-competitive inhibitor. The free enzyme binds phosphate, Mg2+ and fluoride.

The fluoride ions replace the oxygens of carboxylate of PEP.

Question 21

What effect does non-competitive inhibition have on Km and Vmax?

Answer 21

It reduces Vmax but does not affect Km.

It does not affect Km because it is not competing with the substrate for the active site. However, as you increase the concentration of the inhibitor, then you progressively decrease the Vmax of the reaction.

Question 22

What effect does un-competitive inhibition have on Km and Vmax?

Answer 22

It reduces both Km and Vmax.

The inhibitor binds only to the enzyme-substrate complex. The Km is reduces as the complex is depleted and the enzyme and substrate to enzyme-substrate complex equilibrium is restored.

Question 23

Where would we find physiological examples of un-competitive inhibition?

Answer 23

Uncompetitive mechanisms are involved with certain types of cancer.

It has also been found that a number of the genes that code for human alkaline phosphatases (TSAPs) are inhibited uncompetitively by amino acids such as leucine and phenylalanine.

Question 24

What is allosteric regulation?

Answer 24

Allosteric regulation, broadly speaking, is just any form of regulation where the regulatory molecule (an activator or inhibitor) binds to an enzyme someplace other than the active site.

Question 25

How do allosteric enzymes work?

Answer 25

Allosteric enzymes typically have multiple active sites located on different protein subunits.

When an allosteric inhibitor binds to an enzyme, all active sites on the protein subunits are changed slightly so that they work less well.

There are also allosteric activators. Some allosteric activators bind to locations on an enzyme other than the active site, causing an increase in the function of the active site.

Also, in a process called cooperativity, the substrate itself can serve as an allosteric activator: when it binds to one active site, the activity of the other active sites goes up. Cooperativity is considered a allosteric regulation because the substrate affects active sites far from its binding site.

Question 26

Why is allosteric inhibition important physiologically?

Answer 26

Allosteric inhibition is very important (and common) in metabolic pathways because, commonly, an end product of a reaction can act as an inhibitor to the enzyme for that mechanism, acting as a negative feedback loop.

Question 27

Give an example of an allosteric enzyme.

Answer 27

An example of an allosteric enzyme is aspartate transcarbamylase (ATCase).

ATCase catalyses the condensation of aspartate and carbamoyl phosphate to form N-carbamoylaspartate, in pyrimidine synthesis.

Question 28

What is the hallmark of an allosteric enzyme, graphically?

Answer 28

The hallmark of an allosteric enzyme is the presence of a sigmoidal curve in the plot of velocity against substrate concentration.

This is irrespective of negative or positive cooperation.

The sigmoidal response is retained but shifted by regulators. A shift to the left is activation, whilst a shift to the right is inhibition.