Enzymes

Question 1

Are all enzymes proteins?

Answer 1

No, also have catalytic RNAs

Question 2

How do enzymes work?

Answer 2

Reaction rate is restricted due to activation energy. To increase the number of substrates with free energy, you need to heat to provide kinetic energy or increase the concentration of the molecules. Reactions have to go through the transition state, which the higher free energy state in the substrates to a lower free energy state in the products (difference = Gibb’s free energy). Enzymes enhance the reaction rate by decreasing the size of the enzyme activation energy into smaller stages. Enzymes also stabilise transition states.

Question 3

What does attachment of the substrate to the enzyme ensure?

Answer 3

The specificity of the reaction which is catalysed.

Question 4

Purpose of enzyme cofactors?

Answer 4

Can execute chemical reactions which can’t be carried out by amino acids.

Question 5

When can reactions occur spontaneously?

Answer 5

If the reaction is exergonic. and the free energy of the products is lower than the energy of substrates. Can be altered by changing concentrations of substrates.

Question 6

What impact do enzymes have on reaction rate?

Answer 6

Between 10^6 and 10^17 times faster.

Question 7

Michaelis Menten Equations?

Answer 7

Question 8

Assumptions made in Michaelis Menten Equations?

Answer 8

• substrate binding is fast, catalysis is slower
• initial velocity is measured, where [P]=0, therefore reverse reaction can be ignored
• [ES] is constant
• [S] >> [E]_total so [S] = [S]_total
• Enzyme exists in two forms [E] or [ES], and total amount is constant

Question 9

Significance of the Michaelis Menten constants?

Answer 9

Vmax:
V_max = k3[E]_total
-proportional to concentration of enzyme
-max rate of an enzyme catalysed reaction
-k3 is the turnover number which is a measure of enzyme effectiveness
-k3 often referred to as k_cat (catalytic constant) and it is the rate of ES -> P

Km:

• concentration of substrate at which the rate is half Vmax
• measure of the affinity of the enzyme for its substrate (if high then the affinity is low)

Question 10

What is the enzyme specificity factor?

Answer 10

Rate of change between ES>E+S / Km

Question 11

What is kinetic perfection?

Answer 11

Enzyme rate is limited by rate of diffusion into the active site – kinetic perfection

Partially overcome by confining substrates and products in a limited volume of multi-enzyme complex

Allosteric enzymes don’t follow these kinetics. They have multiple subunits and active sites, and the binding of one substrate to an active site can alter properties of other active sites in the same enzyme molecule

Question 12

How does the enzyme-substrate complex form?

Answer 12

Via the induced fit model, where the interaction causes complementarity.

Catalysis is promoted by optimising enzyme-substrate interactions in the transition state. It forces the substrate into a more reactive state, due to the enzyme being complementary to the transition state.

If the enzyme was complementary to the substrate it would make the substrate more stable. The binding energy released in formation of transition state contributes to activation energy.

Question 13

How does the binding of substrates to enzymes speed up reactions?

Answer 13

1. Weakening bonds in substrates
2. Stabilising the transition state
3. Increasing local concentration of substrates in the correct orientation
4. Altering the micro environment
5. Converting complex reactions into a series of bimolecular reactions

Question 14

General principles of enzyme catalysis?

Answer 14

1. Covalent catalysis – at some point the enzyme and substrate are covalently combined
2. General acid-base catalysis – a molecule is used as proton donor/acceptor
3. Metal ion catalysis
   - Electrophilic catalyst (stabilises –ve charge)
   - Generation of nucleophile (promote acidity of nearest molecule)
   - Co-substrate (increase binding interactions)
4. Catalysis by enhanced proximity – bringing reacting molecules together

Question 15

Example of an enzyme that cleaves a strong bond?

Answer 15

Chymotrypsin: cleaving a strong bond

Catalyses the cleavage of peptide bonds. Chymotrypsin can be inactivated through chemical modification of a reactive serine residue.

The serine is part of a catalytic triad, which closely interact with each other in the enzmye. The role of the catlytic triad has been established by site-directed mutagenesis.

Catalytic specificity is provied by a hydrophobic packet.

A two step process involving a covalently bound intermediate.

Question 16

Example of an enzyme that makes a fast reaction faster?

Answer 16

Carbonic anhydrase

Catalyses the hydration of carbon dioxide, by adding water. Contains a bound zinc ion which is essential for catalytic activity.

Binding creates a more acidic environment in the water.

Question 17

Example of an enzyme that avoids the wrong reaction?

Answer 17

Nucleotide monophosphate kinase.

Catalyses the exchange of a phosphoryl group between nucleotides. Many NMP kinases share a common structure - a P loop, which interacts with the terminal phosphoryl groups of ATP.

ATP binding induces a large conformational change in adenylate kinase.

Question 18

Reversible inhibition of enzyme activity?

Answer 18

Competitive inhibitor is a similar shape to the substrate and binds to the enzyme’s active site.

Example: a structural analogue of tetrahydrofolate is a competitive inhibitor of dihydrofolate reductase.

Question 19

Non competitive inhibition?

Answer 19

Non-competitive inhibition is usually also reversible but involves binding to an allosteric site away from the active site on the enzyme, this alters the shape of the active site and prevents the substrate from binding.

If the covalent bonds are strong, this could be irreversible.

Changing the concentration of the substrate doesn’t affect the rate of reaction.

Question 20

What is the cooperative enzyme model?

Answer 20

Tense and relaxed states. Describes allosteric transitions of proteins made up of identical subunits.

Question 21

What is the sequential enzyme model?

Answer 21

A protein’s conformation changes with each binding of a ligand, sequentially changing its affinity for the ligand at neighbouring binding sites.

Ligand binding may also result in a negative cooperativity, a reduced affinity for the ligand – this is the difference between the two.

Sigmoidal kinetics result from a combination of co-operative and sequential interactions in multi-subunit proteins

Question 22

Allosteric modulation and feedback regulation in ATCase and PFK?

Answer 22

ATCase activity is influenced by availability of both CTP & ATP.

One of the products ultimately produces CTP, which then inhibits ATCase – feedback inhibition.

ATP activates the reaction as it shows an imbalance in the types of bases available. ATP high signals there is high energy in the cell so CTP production would be good for DNA production & cell division.

PFK catalyses the entry of hexose phosphate into glycolysis. Uses ATP to breakdown in the first step. ATP binds to the regulatory allosteric site. Displays sigmoidal kinetics.

The reaction is faster in a lower ATP concentration, as it is allosterically inhibited by ATP – need less glycolysis if there is a higher concentration of ATP.

Question 23

How does protein de/phosphorylation control enzyme activity?

Answer 23

• structural: adds 2 –ve charges for electrostatic interactions, can form 3 or 4 H bonds
• thermodynamic: large change in energy for phosphorylation
• kinetic: between 1 second and 1 hour, adjut to meet timing needed for process

Both phosphorylation and dephosphorylation are thermodynamically favourable and regulated.

Question 24

How does reversible covalent modification work?

Answer 24

Glycogen is a branched polymer of glucose units.

Glycogen phosphorylase catalyses the release of glucose units from the non reducing ends of glycogen chains.

Some of the energy from the glyosidic bond is contained in the glucose-1-phosphate which is released from the glycogen.

Question 25

Tense and relaxed states in enzymes?

Answer 25

Phosphorylase has relaxed and tense states due to the subunits being able to rotate relative to each other. In the relaxed form, active site is clear.

In muscle, phosphorylase b is sensitive to allosteric regulation in response to respiratory demand in the cell. ATP causes the enzyme to convert to the tense state. Glucose breakdown isn’t needed when there is already lots of available energy.

In liver, phosphorylase a is regulated by the steady-state glucose level. Glucose converts the enzyme to the tense state. Breakdown isn’t needed when there is already lots of glucose.

Question 26

Activation of enzymes by proteolytic processing?

Answer 26

Chymotrypsinogen.

Proteolysis is the breakdown of proteins into smaller polypeptides or amino acids.

Zymogen: inactive substance converted into an enzyme when activated by another enzyme.

Inactive Chymotrypsinogen (along with other zymogens) are synthesised & secreted by acinar cells in the pancreas.

Trypsin converts Chymotrypsinogen into its active form by cleaving it and removing two dipeptides. Leads to formation of the substrate-binding site. Chymotrypsin activation is part of zymogen activation cascade initiated by enteropeptidase.

Trypsin is protected against premature activation by a tight-binding competitive inhibitor, the inhibitor is cleaved slowly by trypsin but the half-life of bonds is months.