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Flashcards in Enzymes Deck (37):
1

What is an enzyme?

• Globular protein
• Biological catalyst that differs from a chemical catalyst in that they;
– Catalyses very high reaction rates
– Shows great reaction specificity
– Work in mild temperature/pH conditions
– Can be regulated
• Very important – nearly all biochemical reactions involve them

2

What is a ribozyme?

Some enzymes are termed ribozymes – catalytic RNA molecules with no protein component

3

What is a cofactor?

Non-protein component needed for activity
E.g. Fe2+ or Fe3+, K+ and Mg2+

4

What is a coenzyme?

Complex organic molecule, usually formed from vitamins
E.g. FAD, NAD+, Coenzyme A

5

What is a prosthetic group?

Cofactor covalently bound to the enzyme or very tightly associated with the enzyme

6

What is a apoenzyme?

The protein component of an enzyme that contains a cofactor

7

What is a holoenzyme?

"whole enzyme” – the apoenzyme plus the cofactor(s)

8

What is a substrate?

Molecule acted on by the enzyme

9

What is the active site on an enzyme?

Part of the enzyme in which the substrate binds and is acted upon

10

Name the 6 classes of enzymes given by the IUBMB

Oxidoreductases
Transferases
Hydrolases
Isomerases
Lyases
Ligases

Enzymes are OTHILL

11

How to enzymes affect reactions?

They do NOT:
– Move reaction equilibria
– Make a non-spontaneous reaction spontaneous

They DO:
– Increase rates of spontaneous reactions
– Lower the activation energy of biochemical reactions
– Accelerate movement towards reaction equilibrium

12

What is free energy and how can it be calculated?

“Useful” energy generated from cellular reactions is termed Gibbs Free-Energy (G), originally called “available energy”.
Spontaneous reactions must have a –ve ΔG value as they will decrease enthalpy (H) and/or increase entropy (S)

ΔG = ΔH - TΔS

13

How do enzymes reduce activation energy?

• Entropy reduction, physically unite molecules
• Desolvation, replaces bonds between water and substrate
• Induced fit

14

How can you scientifically analyse an enzyme?

o Enzyme kinetics – information on substrate and product affinity to the enzyme, knowledge of the dynamic properties of enzyme catalysis is useful in drug design
o Mutagenesis – useful in the study of enzyme effects, identification of the active site, and the design of novel proteins
o 3D structure – good for identifying structure, function if uncharacterized, interactions with other macromolecules, structure based drug design etc.

15

Define the ‘Michaelis Constant’, describe how it may be experimentally estimated and describe what knowledge of the constant may allow to be deduced about the nature of the catalysed reaction.

Km = ½ Vmax = k-1 + k2 / k1

Larger Km values indicate a less stable ES complex.
Lower Km values indicate a more stable ES complex.

Vmax also tells us the degree of activity of an enzyme.

Therefore, it tells us about the affinity of the enzyme for the substrate.

16

What does Km represent?

“Km is equivalent to the substrate concentration at which the initial reaction rate is half of the maximum reaction rate”

17

What are isoenzymes?

Different enzymes that catalyse the same reaction.

E.g. hexokinase (muscle) and glucokinase (liver; aka hexokinase D) both phosphorylate glucose to G6P, but have different Km for glucose

18

What is gel electrophoresis?

Gel electrophoresis is a laboratory method used to separate mixtures of DNA, RNA, or proteins according to molecular size.
DNA is negatively charged, therefore, when an electric current is applied to the gel, DNA will migrate towards the positively charged electrode. Shorter strands of DNA move more quickly through the gel than longer strands resulting in the fragments being arranged in order of size.

19

Whats an allosteric enzyme?

Allosteric enzymes are enzymes that change their conformational ensemble upon binding of an effector, which results in an apparent change in binding affinity at a different ligand binding site.

20

What can affect enzyme function?

Temperature
pH
Inhibitors

21

What is a competitive inhibitor?

• These bind to enzymes non-covalently and will usually resemble the substrate molecule, therefore competing with the active site
• This leads to a decrease in the affinity between the active site and the substrate, so the Km of the substrate-enzyme complex increases
• Increasing substrate concentration can overcome this inhibition, so the same Vmax can be achieved

22

What is a non-competitive inhibitor?

• These bind to enzymes non-covalently and will usually attach to a site other than the active site of the enzyme
• The substrate is usually still able to bind the active site, so the Km of the substrate-enzyme complex remains unchanged
• Increasing substrate concentration does not change the inhibition so the Vmax will decrease

23

What is an irreversible inhibitor?

• This type of inhibitor will usually bind to the enzyme in a covalent, and therefore irreversible way

24

Describe the two models that explain allosteric enzyme kinetics

The concerted model
The sequential method

25

Describe the concerted model

The concerted model
• Each subunit can exist in two different conformations, where one binds the substrate well while the other doesn’t
• With no substrate, the enzyme flips between the two conformations
• All subunits must be in the same conformation (flip in concert)
• When one molecule of S binds, this ‘locks’ the other binding sites, stopping them flipping, allowing other S to bind easily

26

Describe the sequential model

This model assumes:
– No flipping between different conformation states
– Sub-units exist in a conformation that can bind S, activators, inhibitors
– It is the binding that causes a conformational change
Substrate binding causes a change in one sub-unit. This causes a change in another sub-unit allowing it to bind S more readily

27

How can enzymes be modulated?

– Allosterically
– Covalent modifications
– Proteolytic cleavage

28

What are the four methods that inhibitors can affect enzymes?

– Competitive inhibitor
– Non-competitive inhibitor
– Irreversible inhibitor
– Feedback inhibition

29

How is the rate of the reaction affected by changes in concentration of enzyme or the substrate.

Normally in a reaction, as substrate concentration increases the rate of the reaction does increase until all of the enzymes binding spaces are utilised, resulting in a plateau at Vmax.

If enzyme concentrations were to be increased this could result in a higher Vmax, though substrate concentration may be a limiting factor if the enzyme concentration is increased too dramatically.

30

What enzyme assays are used in clinical diagnosis?

Gel electrophoresis e.g. elevated CK2 indicates MI
Measure activity
Presence of intracellular enzymes in blood - damage

31

Why is the Lineweaver-Burk pet better than the M-M model?

More accurate reading of Vmax from linear graph, and also therefore Km reading

32

What enzyme is found elevated in the plasma of those who have recently had a myocardial infarction?

CK2 (heterozygous BM isoform of creatine kinase)

33

Whats the difference between uncompetitive and non-competitive inhibitors?

Uncomp - binds only ES complex at allosteric site
Non-comp - binds E or ES at allosteric site

34

Describe the action of Azidothymidine

Competitive inhibitor of reverse transcriptase
Used to treat retroviral HIV infections

35

Describe the action of Oseltamivir

Transition analogue (max interaction of enzymes are with transition state molecules)
Hydrolysed in body so it can block neuraminidase
This normally cleaves sialic acid to allow release of virus
For influenza A and B infections

36

Describe a natural catalytic antibody

Those of lupus erythematosus - autoantibodies attack connective tissue of joints, skin, heart and lungs

37

Describe the most common type of covalent modification of enzymes

Phosphorylation and dephosphorylation