Enzymes as drug targets

Question 1

How do most drugs work?

Answer 1

Drugs normally contain inhibiting mechanism which prevents the enzymes from catalyzing biological reactions.
Without these enzymes available the reaction cannot take place.

Question 2

Where are enzymes usually found in the cell?

Answer 2

Protein enzymes are usually found either in the soluble cytoplasmic form or membrane bound.

Question 3

Which properties do enzymes affect?

Answer 3

Enzymes do affect kinetic properties. For example, enzymes catalyses reactions by lowering the activation energy (energy difference between the substrate and the transition state). However enzymes do not affect thermodynamic properties; they don’t alter the Gibbs free energy (the energy difference between the substrate and products).

Question 4

What is the overall equation that depicts the substrate binding process?

Answer 4

S + E [ES] E + P

Question 5

How is a substrate able to bind to a protein-enzyme?

Answer 5

Just as with receptors the amino acid side chains must form complimentary binding interactions with the residues on the enzyme.
The substrate must also be the correct size and shape in order to fit into the active site as well as the correct orientation.

Question 6

Describe the substrate binding process.

Answer 6

The substrate (providing it has the correct size, shape, residues and orientation) binds to the active site on the enzyme forming an enzyme-substrate complex. 
The binding of the substrate to the active site pulls on the stress of the chemical bond within the substrate causing it to break. 
The products are now formed.

Question 7

How do protein catalysts lower the activation energy?

Answer 7

Essentially the enzymes wrap themselves around the substrate which stabilizes the transition state (high energy state). By stabilizing it, in turn its energy is lowered.

Question 8

What is the difference in enzymatic activity of synthases and synthetases?

Answer 8

Synthases: condenses two molecules together
Synthetases: condense two molecules together and ATP is used to drive the reaction.

Question 9

What is the function of isomerases?

Answer 9

Rearrange bonds within a molecule

Question 10

What is the main drug target of most cancer treatments?

Answer 10

Most cancer treatments contain enzymatic inhibition of kinase activity. Kinases add a phosphate group to proteins which activates downstream effects in intracelluar signalling pathways. When the reverse mechanism is not not functioning over activation of kinase activity can cause cancer, therefore cancer treatments are targeted to inhibit the kinase activity.

Question 11

Function of DNA polymerase?

Answer 11

Catalyses DNA polymerisation reactions

Question 12

List the four main enzyme-substrate interactions (increasing to decreasing strength).

Answer 12

Covalent interactions
Ionic interactions
Hydrogen bond interactions
Hydrophobic interactions

Question 13

What are co-factors?

Answer 13

Co factors are the addition of a non-protein which associates close to the enzyme binding site to help facilitate the binding of the substrate to the enzymes.

Question 14

Describe the different types of co-factors.

Answer 14

Can have organic and inorganic co-factors which can be further subdivided into whether they are permanently associated with the active site (carboxylases) or whether they are loosely associated (NAD+).

Question 15

What is the difference between Haloenzyme and Apoenzyme?

Answer 15

Haloenzyme: protein part of enzyme + cofactor
Apoenzyme: protein part of the enzyme - cofactor

Question 16

Names for dissociable and non-dissociable cofactors?

Answer 16

Co-enzymes: dissociable

Prosthetic group: non-dissociable

Question 17

When does induced fit occur?

Answer 17

It occurs for enzymes that are less specific.

Question 18

Define KM?

Answer 18

Km is the concentration of enzyme required to produce half of the vmax (maximum rate of reaction).

Question 19

How is the km found graphically?

Answer 19

If you plot a graph of V0 (rate of reaction, y axis) against [S] (substrate concentration, x axis), draw a line across horizontally for the max y value which gives the vmax (e.g 10) . From that half the y value (e.g 5) and then go across and find the corresponding x value. This is km.

Question 20

What does the curve for rate of reaction vs substrate concentration usually look like?

Answer 20

It is a curve shaped beginning at (0,0). As the substrate concentration begins to increase, quite quickly the rate of reaction also increases (steep gradient). However the gradient is always decreasing as the enzymes begin to become saturated; the curve flattens out. At a certain point no matter how much the concentration of substrate increases, the rate will no longer increase, enzymes are fully saturated.

Question 21

What is the Lineweaver- Burke plot?

Answer 21

It is the same graph ( rate of reaction vs substrate conc. but instead the reciprocals are taken for each which makes the graph linear.

Question 22

What are the key points of the Lineweaver-Burke plot?

Answer 22

Gradient: km/v max

y-intercept: 1/ v max

x-intercept: -1/ km

Question 23

What are the two factors that affect substrate efficency?

Answer 23

Km: the substrate concentration that is required for the reaction rate to be half of the maximum.

Kcat: the number of substrate molecules converted to products per enzyme per second.

Question 24

What is the equation for catalytic efficiency?

Answer 24

Catalytic efficiency = kcat/km

Question 25

What is the optimum catalytic properties?

Answer 25

E + S [ES] —-> E + P

Ideally you want a low km ( E + S [ES] ) as this means a low concentration of substrate is required to produce 50% of the max rate of reaction, so it has a high binding affinity.
However ideally you want a high kcat, so there is a high rate of catalysation (conversion of complex to products).

Question 26

How is rate of reaction determined by proximity?

Answer 26

As a template by the enzyme bringing the two reactants close together as they are attached in a aliphatic ring. The smaller the ring the faster the rate of reaction.

Question 27

Which amino acids form covalent interactions in enzymes?

Answer 27

Serine and cysteine both have nucleophilic properties which enables them to covalent bonds with the substrate.

Question 28

Explain electrostatic catalysis?

Answer 28

The enzyme uses an ionic charge or partial ionic charge to interact with an opposite charge developing on the substrate at the transition state of the reaction.

Question 29

What is desolvation?

Answer 29

Desolvation is when the ground state of the enzyme is destablised by removing the water molecules so that hydrophobic interactions can form.

Question 30

How does strain distort the ground state of the substrate?

Answer 30

When the substrate is put under strain it twists and rotates the bonds.

Question 31

When does denaturing occur?

Answer 31

The binding interactions that holds the enzyme together break above a certain temperature causing the shape of the active site to change.

Question 32

How does pH affect the enzymatic activity?

Answer 32

The active site of the enzyme might have an aspartic acid or glutamic acid amino acid residue that requires deprotonation to form electrostatic interactions with the substrate. If this enzyme is in acidic conditions then this deprotonation will not occur.

Question 33

Describe transition state analoges.

Answer 33

Transition state analoges are inhibitors that mimic the structure of the substrate in its transition step so forms the same strong interactions with the enzyme in the active site.

Question 34

What are the key differences between competitive and non-competitive enzyme inhibitors.

Answer 34

Competitive inhibitors bind to the substrate active site, effectively blocking the substrate out.
The km is reduced (the affinity).

Non-competitive inhibitors bind to a different part of the enzyme, different to the substrate binding site, however they work by reducing the activity of the enzyme.
Vmax is reduced.

Question 35

How can you tell graphically whether an inhibitor is competitive or non-competitive?

Answer 35

If it is competitive on a graph with a enzyme with no inhibitor present, the lines will cross on the y axis.

If it is non-competitive on a graph with a enzyme with no inhibitor present, the lines will cross on the x axis.

Question 36

Give examples of competitive and non-competitive inhibitors.

Answer 36

Competitive: Methotrexate, a chemotherapeutic drug that binds to dihydrofolate reductase enzyme.

Non-competitive: Aspirin bind to cyclo-oxygenase.

Question 37

How do you test for inhibition?

Answer 37

Step 1: Take enzyme with substrate at a fixed concentration and record the rate. Then add an inhibitor.

Step 2: Measure the IC50
Repeat Step 1 but with inhibitors at different concentrations. Need about 8 to 10 concentrations.

Question 38

How do you test for the mode of inhibition?

Answer 38

This is assessing whether something is a competitive or non-competitive enzyme.
To do this you need to analyse the relationship between the changing concentration of the inhibitor and the rate of the reaction.
Firstly record data for rate vs substrate without an inhibitor present and then one with an inhibitor.
Plot a rate vs substrate concentration (reciprocals) and from that determine whether the lines cross on the y or x axis indicating whether the inhibitor is competitive or non-competitive.

Question 39

What are proteases?

Answer 39

They are the enzymes that are responsible for hydrolysing the amide bond found in polypeptide chains.

Question 40

What are some of the biological roles of proteases?

Answer 40

They break down protein content found in food and digest it.
Involved in cell signalling
Helps initiate blood clotting (thrombin)

Question 41

Explain what is meant by a peptidomimetic.

Answer 41

They are a non-peptide drug used to mimic the action of a peptide.

Question 42

What are the four advantages of peptidomimetics? (2is, 2rs)

Answer 42

Increase bioavailability
Increase stability to digestive/metabolic enzymes
Reduced cost
Reduced antigenicity

Question 43

How can you convert a peptide to peptidomimetic?

Answer 43

Bioisosteric replacement of the amide link.
Could convert an amide to an alkene, kentone, amine etc.

Replacement of a natural amino acid with a non-natural one.

Alter the stereochemistry: replacement of l amino acids with the d-enantiomers.

Question 44

What is the difference between L and D enantiomers?

Answer 44

L isomers have the hydroxy group attached to the left side of the asymmetric carbon furthest from the carbonyl (now called S) while D isomers have the hydroxy group on the right side (R).

Question 45

Which amino acid residues are responsible for 1 step catalysis?

Answer 45

Aspartic acid protases, Glutamic acid proteases and metallo proteases.

Question 46

Describe the catalysis that Serine, Threonine and Cysteine proteases perform?

Answer 46

Each have a nucelophilic residue on the amino acid which attacks the electron deficient carbonyl which in return cleaves the C-N amide bond. The carbonyl carbon is then attacked by water, as the bond between the nucleophilic residue and substrate breaks. The enzyme is regnerated.

Question 47

Describe the mechanism undergo by aspartyl protease.

Answer 47

Aspartyl protease contains an carboxylic acid which activates a water molecule which then attacks the electron deficient carbonyl carbon; cleaving the amide bond.

Question 48

What is Saquinavir?

Answer 48

It is an antiretroviral protease inhibitor that is used in therapy and the prevention of HIV.

Question 49

How do protease inhibitors work?

Answer 49

They act by binding to the viral protease, preventing the cleavage of viral proteins.

Question 50

Key geographically differences between HIV-1 and HIV-2?

Answer 50

HIV-1 is the most widespread and is 95% of all HIV cases. HIV-2 is found in Western Africa.
HIV-2 is less fatal and progresses more slowly than HIV-1.

Question 51

How do competitive inhibitors work?

Answer 51

Competitive inhibitors work by forming covalent bonds. The covalent bonds form when a nucleophilic amino acid residue on the side chain of the enzymes aligns perfectly with an electrophilic functional group on the substrate. Once the nucleophile and electrophile interact a permanent covalent bond is formed.

Question 52

What determines the speed of the formation of the permanent covalent interaction?

Answer 52

The relative position of the electrophile and nucleophile to each other. If they are perfectly aligned the covalent bonds can form within seconds; if they are further apart it can take up to hours.

Question 53

What are some examples of amino acids that have nucleophilic residues?

Answer 53

Containing:

• OH: Serine, Threonine
• NH2: Arginine, Lysine, Asparagine, Glutamine
• SH: Cysteine
• COO(-): Aspartic acid and Glutamic acid

Question 54

How are Aspartic and Glutamic acid able to undergo nucleophilic attack?

Answer 54

When deprotonated the carboxylate anions act as weak nuleophiles. When perfectly aligned and a strong electrophile is present they can acts as nuclophiles and attack the electron deficient carbon.

Question 55

List examples of electrophiles present on inhibitors.

Answer 55

Alkyl halides
Amides
Esters
Michael Acceptors
Epoxides

Question 56

How are competitive inhibitors developed?

Answer 56

The enzyme and its natural substrate are studied are a portion of the peptide chain that binds in the active site it inspected. Essentially one of the amide bonds is replaced with a electrophile so that a covalent bond can form between the drug molecule and enzyme.

Question 57

Describe the key structural differences between a non-competitive inhibitor and a competitive inhibitor graph?

Answer 57

In a graph that plots the concentration of product (y-axis) against time ( x axis); for a non-competitive inhibitor in comparison to when a inhibitor is not present it is just a lower but linear gradient.
Whereas with a competitive inhibitor the line is curved (decreasing gradient) which then flattens out as the enzyme stops working altogether.

Question 58

Explain the curvature of the competitive enzyme, conc. vs time graph?

Answer 58

EI* E ES —> P

This is the equilibrium that is formed.
With a competitive inhibitor the concentration of EI* (the covalently bonded enzyme and inhibitor complex) although may take hours to form, decreases the conc. of EI which causes equilibrium to shift to the left, decreasing the conc. of E. Equilibrium shifts to the left again, decreasing the conc. of ES. Eventually no product can be formed as ES is depleted completely.

Question 59

What are the advantages and disadvantages of competitive inhibitors?

Answer 59

Advantages:
Causes permanent enzyme inhibition
A high conc. of the inhibitor is not required

Disadvantages:
Specificity
Electrophile stability

Question 60

Why are rates of penicillin allergy so high?

Answer 60

The lack of specificity of the nucleophile that binds means there is a lot of background activity in addition to attack of the four membered ring. The greater the background activity, the greater the possibility of side effects.

Question 61

What is a catalytic triad?

Answer 61

A catalytic triad is a set of three co-ordinated amino acids found in the active site of some enzymes.

Question 62

What is the most common catalytic triad and how does it help promote its function?

Answer 62

The most common catalytic triad is the acid-base-nucleophile triad which through a charge relay network helps to polarise, deprotonate and activate the nucleophile found in serine, threonine and cysteine residues which enables them to undergo nulceophilic attack forming a covalent bond.

Question 63

What are some covalent inhibitors of cysteine, serine and threonine proteases?

Answer 63

Salinosporamide
Licochalcone A
E-64

Question 64

How do zinc metallo-proteases work?

Answer 64

A zinc metal is co-ordinated into an amino acid side chain of the enzyme. When a nucleophilic residue attacks the electron deficient carbonyl, electrons are pushed onto the oxygen resulting in it having a negative charge. This negative charge is then stabilised with the positive charge of the metal ion.