Proteins and Enzymes

Question 1

What are the seven possible functions of proteins?

Answer 1

Enzymes

Bind molecules for storage and support

Provide support and structure

Do mechanical work

Decode cell information

Hormones

Other specialised functions (eg antibodies)

Question 2

What are the 4 major functional groups of proteins?

Answer 2

Binding

Catalysis

Switching

Structural

Question 3

What is pKa?

Answer 3

The pH at which a group capable of dissociation is 50% dissociated

Question 4

How do groups behave at different pHs?

Answer 4

pH > pKa → group is always negatively charged

pH < pKa → group is always positively charged

Question 5

What shape amino acid stereoisomer is predominant in nature?

Answer 5

L-amino acids

Question 6

Which amino acids have aliphatic R groups, and what are their properties?

Answer 6

Glycine → imparts structural flexibility

Alanine

Valine

Leucine

Isoleucine → most hydrophobic; has 2 chiral centres so can form 4 stereoisomers

Question 7

Which amino acids have acidic R groups (or their amide derivatives), and what are their properties?

Answer 7

Aspartate → gives proteins negative charges; ionised at pH 7

Glutamate→ gives proteins negative charges; ionised at pH 7

Asparagine → highly polar

Glutamine → highly polar

All can form H bonds

All can act as bases

Question 8

Which amino acids have basic R groups, and what are their properties?

Answer 8

Histidine → can act as an acid and a base due to lone pair on N and protonated N; can bind metal ions

Lysine → positively charged at physiological pH; contributes basicity to proteins

Arginine → most basic; positively charged at physiological pH

All have very high pKas so Lys and Arg are positively charged at pH7.

Often involved in ion pairs.

Question 9

Which amino acids have alcohol or sulphur groups, and what are are their properties?

Answer 9

Methionine → has a protected thiol group (non-polar methyl thioether group); hydrophobic

Cysteine → somewhat hydrophobic; extremely reactive; can form disulphide bonds making the proteins more stable

Serine → doesn’t ionise; hydrophilic; can be phosphorylated due to OH group

Threonine → 4 stereoisomers; can be phosphorylated due to OH group

Question 10

Which amino acids have nitrogen heterocycle R groups, and what are their properties?

Answer 10

Proline → unique structure; helix breaker; can exist in cis and trans state

Phenylalanine → hydrophobic; usually on the inside

Tyrosine → hydrophobic; usually on the inside; may be on the outside due to OH group (ring enhances stability of conjugate base); can sometimes act as an acid

Tryptophan → hydrophobic; usually on the inside

Question 11

What is the shape of the peptide bond? What rotations are allowed?

Answer 11

The nitrogen of the peptide bond forms a trigonal planar shape due to one resonance form having a double bond between the N and C.

The peptide bond is, therefore, planar and cannot rotate.

Peptide bonds can rotate relative to each other because the bonds around the peptide bond can rotate.

These are very flexible and allow hydrophobic side chains to be sequestered away from water.

Question 12

What is a Ramachandran plot?

Answer 12

A diagram to show the degree of rotation of the bonds next to the peptide bond.

It, therefore, shows which side chains are most flexible.

It was developed by G. N. Ramachandran et al

Question 13

What is the primary structure of a protein?

Answer 13

The amino acid sequence of a protein, dictated by the genetic code.

This sequence contains all the information needed to specify the secondary, tertiary, and if applicable, the quaternary structure.

Question 14

What is the secondary structure of a protein?

Answer 14

The regular repeating patterns of H-bonded backbone conformations, such as α​-helix and β-sheets.​

The H-bonding only occurs between the carbonyl O and amide N of two peptide bonds.

Question 15

What is the tertiary structure of a protein?

Answer 15

How the secondary structural elements pack together to form the overall shape of the protein in the form of folds.

These are mostly non-covalent, R-group interactions.

Question 16

What is the quaternary structure of a protein?

Answer 16

The overall relative arrangement of two or more individual tertiary folded polypeptides.

Only proteins made up of multiple subunits will have a quaternary structure.

This is always due to non-covalent interactions, which gives the protein flexibility.

Question 17

Do polypeptides have the cis or trans conformation, and why?

Answer 17

The cis conformation usually results in too much chemical clash.

The trans conformation has the R groups on alternating sides, which avoids this problem.

Question 18

What is the chemical reactivity of hydrophobic, hydrophilic and amphipathic side chains?

Answer 18

Hydrophobic → engage in Van de Waals interactions. They avoid contact with water, so pack together, forming the basis of the hydrophobic effect

Hydrophilic → can form H-bonds with each other, the peptide bond, other polar molecules and water. Some side chains will be charged, so this will change depending on the pH.

Amphipathic → have both polar and non-polar character. These are ideal at interfaces, and may be involved in both Van de Waals and H-bonding.

Question 19

What are the three general types of secondary structure?

Answer 19

Helices → the most common is the alpha helix

Beta sheets → sometimes known as pleasted sheets. They can exist as parallel or anti-parallel

Beta turns → the chain is forced to turn sharply in a reverse direction; this small secondary structural element allows for the compact folding of proteins

Question 20

How does H-bonding form an alpha helix?

Answer 20

The carbonyl oxygen atom (n) accepts an H-bond from the amide nitrogen four residues further along (n+4)

All of the polar amide groups of this helix are H-bonded to each other, except the first amide hydrogen and the last carbonyl oxygen

Question 21

Where are the R groups on an alpha helix?

Answer 21

The walls of the helix are formed by the H-bonded backbone with the side chains pointing outwards.

Under some circumstances the helix can be amphipathic, with a hydrophic nature on one side and hydrophilic nature on the other.

Question 22

What are the general properties of alpha helices?

Answer 22

They can be right-handed or left-handed, but right-handedness is usually favoured due to steric factors

There is no theoretical limit to the length of the helix

There are variants of the alpha helix but these are not common

Proline is a helix breaker because it cannot donate an H for H-bonding further up the chain. For this reason, prolines are found at the end of a helices

Question 23

What is the structure of beta sheets?

Answer 23

H-bonds form between the amide hydrogen and the carbonyl oxygen; these come from groups distant from each other in the primary sequence.

There is no pattern for peptide H-bonding.

Two or more strands lay side-by-side, with H-bonding between the strands.

The strands can run in the same direction (both 5’ - 3’, parallel) or in opposite directions (one 5’ - 3’, the other 3’ to 5’, antiparallel)

H-bonds are more linear in the antiparallel sheet.

Question 24

What are the general properties of beta sheets?

Answer 24

Nearly all polar amide groups are H-bonded to one another in a sheet structure

The N-H and C=O groups on the outer edges and edge strands are not h-bonded to other strand members; instead, they can H-bond to water, or may pack against polar side chains (perhaps in a nearby helix).

Parallel sheets are always buried in the protein structure; they are usually hydrophobic because sheets can contain bulky side groups, whereas the helix cannot.

Antiparallel sheets are frequently exposed to the solvent and are probably more stable structures (maybe due to more linear H-bonds?)

Parallel beta sheets are always separated by another structural element, usually helices

Beta sheets are nearly fully extended with 3.3Å between residues

Beta strands have a pronounced right-handed twist due to steric factors

Beta strands can be amphipathic due to the alternating consecutive side chain configuration. These strands are found on the surface of proteins.

Question 25

What is the structure of a beta barrel?

Answer 25

A large anti-parallel beta sheet curves all the way around, with the last hydrogen bonded to the first, thus forming a closed cylinder.

Question 26

What is a beta turn?

Answer 26

It compromises of an H-bond from the carbonyl oxygen of one residue (n) to the amide N-H of residue (n+3).

This reverses the direction of the chain.

Beta turns are most commonly found on the surface of proteins in contact with the aqueous environment.

Beta turns have a tight geometry, so require side chains to be small, hydrophilic and polar eg. glycine, alanine, proline

Question 27

What drives protein folding?

Answer 27

Polar/charged R groups and peptide groups will be able to H-bond to water, but non-polar groups cannot and will disrupt the H-bonded structure of nearby water.

To minimise the unfavourable interactions, non-polar R groups tend to clump together; this is the hydrophobic effect. This also brings polarisable hydrophobic groups together, allowing Van de Waals interactions to occur.

Polar backbone amide groups that are dragged into the more hydrophobic interior of the protein satisfy their H-bonds by forming secondary structural elements with other main chain donors and acceptors.

Question 28

What is denaturation, and how does it occur?

Answer 28

A loss in biological activity that is evidenced by the unfolded state.

Heating can break the weak H-bonds that stabilise the native state, leading to conversion to the unfolded state.

Denaturants and detergents compete for H-bonds with polar groups in the chain.

This strips the hydration shell and means the protein is not at its most thermodynamically stable state ie. it is denatured.

Question 29

What interactions stabilise the tertiary structure?

Answer 29

H-bonding, Van de Waals, electrostatic interactions, salt bridges and disulfide bonds.

Disulfide bonds are the only covalent force, which gives the protein extra support.

These interactions only involve R groups.

Co-ordination of a metal ion can occur, which also stabilises the proteins shape.

Cofactor binding can also stabilise the structure, as well as provide chemical reactivity.

Post-translational modification, such as phosphorylation, glycosylation and ubiquitination (addition of ubiquitin, a small regulatory protein), may also change and stabilise the tertiary structure.

Question 30

What are loop regions in proteins, and how are they involved in the tertiary structure?

Answer 30

Loops are long stretches (4+ amino acids) between secondary structural elements.

They are usually found at the surface of the protein and often protrude out into the solvent.

Loops are often involved in the function of the protein, eg ligand/membrane binding or substrate recognition.

They do not contribute much stability so can tolerate mutations more readily; this provides a mechanism for evolution.

Question 31

Why are water molecules important to proteins?

Answer 31

Polar groups on the surface of the protein interact with the water, forming a hydration shell.

Water makes the tertiary structure more stable because the hydration shell exerts pressure on the protein, keeping it in its stable shape.

Some water molecules will be trapped inside the protein; these are a part of the tertiary structure and may be important for the proteins functional activity.

Question 32

How does the tertiary structure allow the protein to be flexible, and why is this useful?

Answer 32

The forces that stabilise the tertiary structure are mostly non-covalent, so they can break and reform easily.

Proteins fluctuate around their equilibrium structure; this can be key to their function eg. enzymes and the induced fit mechanism of substrate binding.

Some areas of the protein are more flexible than others; more flexible areas are involved in the function, whereas more inflexible areas are involved in supporting the protein.

Question 33

Which factors determine the quaternary structure?

Answer 33

Quaternary structure is dependent upon complementarity.

The fit between multiple subunits depends upon many things:

• Shape (which depends upon the primary, secondary and tertiary structures)
• H-bond donors are opposite (and close enough) to acceptors
• Non-polar groups are opposite other non-polar groups
• Positive charges are opposite negative charges

This principle of complementarity is observed in all binding interactions: at interfaces, at binding site for ligands or substrates etc

Question 34

What type of motion in proteins in associated with different functions?

Answer 34

Very fast and small scale movements → catalysis (making/breaking H-bonds)

Mid-range spatial and temporal motions → conformational changes upon binding or signalling

Slow and large-scale motions → binding events

Many of these movements involve the release of bound water and the making/breaking of non-covalent interactions as the protein moves or subunits move relative to each other.

Question 35

Why do binding sites generally have a higher than average amount of exposed hydrophobic groups?

Answer 35

These hydrophobic groups will be buried when the ligand binds; so having these groups exposed encourages the ligand to bind

Question 36

Where does the energy for driving binding events come from?

Answer 36

It is provided by the displacement of water molecules from the ligand binding site.

When the ligand is not bound to the protein, water is bound to the active site.

Question 37

What is the dissociation constant, K_d?

Answer 37

K_d is the molar concentration of ligand at which half of the ligand binding sites are occupied.

K_d is the equilibrium constant for the release of ligand.

When [ligand] is lower than K_d, then very little ligand is bound.

In order for 90% of ligand binding sites to be occupied, [ligand] must be 9 x K_d

The lower the value of K_d, the higher the affinity of the ligand for the protein. This means the ligand is more tightly bound.

Question 38

What is the structure of myoglobin?

Answer 38

Myoglobin has only one subunit.

It is associated with a prosthetic group - the protoporphyrin ring.

The porphyrin ring has 4 coordinate bonds to the Fe²⁺ (Ferrous state) ion

Myoglobin is made up of several helices (A - H) which are linked by loops.

Both the F-helix and E-helix has histidine residues, which sit either side of the porphyrin ring.

The proximal histidine (on F-helix) makes the 5th coordinate bond to Fe²⁺. In the absence of O₂, this causes the porphyrin ring to become puckered.

Question 39

How does oxygen bind to myoglobin?

Answer 39

Oxygen coordinately bonds the Fe²⁺ , which pulls the plane of the porphyrin ring flat.

This makes the oxygen become a radical-like species, so the proximal histidine (on the E-helix) forms an H-bond with the oxygen, stabilising it.

This causes the F-helix to move towards to porphyrin ring, making the protein curl around the oxygen molecule.

Question 40

What are the oxygen binding curves for myoglobin and haemoglobin?

Answer 40

Myoglobin → hyperbolic (reflects that Mb has a high affinity for oxygen, so it is half saturated at low oxygen concentrations)

Haemoglobin → sigmoidal (reflects that Hb is allosteric)

Question 41

What is the structure of haemoglobin?

Answer 41

Hb has a quaternary structure

It is made up of 4 subunits, 2x α and 2x β

Each subunit has a bound haem group containing an iron in the Ferrous state (Fe²⁺), so each can bind a molecule of oxygen

Each subunit is sequentially different to myoglobin but structurally very similar. This means that key residues are highly conserved eg. proximal and distal histidines

Question 42

Who discovered that there are two main forms of haemoglobin?

Answer 42

Max Perutz.

He did this by studying crystallised Hb and Mb

Question 43

How does oxygen bind to haemoglobin?

Answer 43

Hb has two states, tense (T) and relaxed (R). The T state has a low affinity for oxygen while the R state has a high affinity for oxygen.

Binding of oxygen in the T state triggers the subunit to change to the R state.

Oxygen binds to the 6th coordinate position on Fe²⁺ , which pulls the iron into the plane of the porphyrin ring and brings the F-helix closer.

In myoglobin this simply stabilises oxygen binding, but in Hb this causes the subunit to rotate relative to the other subunits, changing it from the T to the R state.

Question 44

How does Hb bind oxygen cooperatively?

Answer 44

Oxygen binding to individual subunits can alter the affinity for oxygen in adjacent subunits.

The first molecule of oxygen that binds to Hb does so weakly because the subunit is in the T state.

This leads to a conformational change that is communicated to adjacent subunits, making it easier for additional oxygen molecules to bind.

T to R transition occurs more readily in the second subunit once oxygen is bound to the first.

Binding of oxygen to the final subunit is high affinity because it is already in the R state.

Question 45

What is an allosteric protein?

Answer 45

Proteins which have other conformations induced by the binding of ligands.

The conformational changes induced by the ligand interconvert more or less active forms of the protein.

In Hb, the binding of one ligand affects the affinities of any remaining unfilled binding sites.

Oxygen activates Hb by causing conformational changes in the first unit that are transferred to the rest of the molecule by subunit to subunit interactions.

Question 46

What roles is ligand binding involved in?

Answer 46

Carrier proteins must bind a ligand

Receptors must bind a ligand to elicit their effect

Switching proteins must bind a trigger molecule, which may cause a conformational change leading to activation/deactivation of another component of the signalling system.

Structural proteins often bind other monomeric proteins of the same type to form polymeric structures

DNA and RNA binding proteins must effectively bind macromolecular structures, or associate with other proteins in large assemblies

Enzymes responsible for dramatic rate enhancements must effectively bind substrates to elicit their effect

Question 47

How do enzymes speed up reaction rates?

Answer 47

Enzymes overcome energy barriers.

Enzymes can hold two very reactive molecules next to each other in the correct orientation; this may be all they need to react without any special chemistry.

Some enzymes bind substrates in a way that destabilises the ground state (raises its free energy). This means the substrate will be less stable, thereby making the passage to product require less energy.

Other enzymes stabilise the transition state (transition state = highest point in the free energy of a reaction pathway) (lowers the free energy), meaning the energy required for the reaction is lowered, so it occurs much faster.

Question 48

Why do enzymes have an optimal pH?

Answer 48

Enzymes work best at a particular pH because the ionisation state of the active site must be correct.

The optimum pH is that at which the binding and catalytic groups are correctly ionised for their functions.

Additionally, the ionisation of R groups must be correct in order for the protein to form its most thermodynamically stable tertiary structure.

Question 49

What type of reactions can enzymes catalyse?

Answer 49

Redox reactions → involve the transfer of electrons

Addition → The addition of atoms or chemical groups to double bonds

Elimination → Removing atoms or chemical groups to form double bonds

Hydrolysis → The cleavage of esters, amides or acetals by reaction with water.

Condensation → The formation of esters, amides and acetals require the removal of water.

Decarboxylation → Removal of a single carbon atom by loss of carbon dioxide.

Question 50

Why do enzymes need cofactors?

Answer 50

There is limited chemical reactivity in the active site of enzymes; acid-base reactions are promoted due to the presence of acidic and basic side chains, as well as histidine.

Enzymes, therefore, have to recruit other molecules to assist in catalysis. These molecules are called cofactors.

Cofactors that are organic compounds and assist in catalytic chemistry are called coenzymes.

Coenzymes can be cosubstrates (weakly bound) or prosthetic groups (tightly bound).

Essential metal ions can be activator ions (weakly bound) or active site ions (tightly bound).

Question 51

What are the 6 types of enzymes?

Answer 51

Hydrolases: AB + H₂O → AH + HO-B

Oxidoreductases (redox reactions): AH₂ + B → A + BH₂

Transferases (kinases are phosphotransferases): AB + C → A + BC

Isomerases: A → isoA

Lyases (splitting): AB → A + B (does not involve water, unlike hydrolases)

Ligases (joining): A + B → AB

Question 52

What are the assumptions (or preconditions) for enzyme kinematics?

Answer 52

Only the initial velocity is being considered, ie. when [P] = 0

[S] >> [E}

This gives a steady [E.S] because the enzyme will have lots of substrate to react with (steady state approximation)

Question 53

What is the Michaelis constant, K_M ?

Answer 53

The molar concentration of substrate at which half of the active sites are occupied.

K_M is a measure of the affinity of the enzyme for its substrate.

A low K_M means a high affinity of the enzyme for the substrate.

Question 54

What is the Michaelis-Menten Relationship?

Answer 54

The relationship between the rate of reaction and substrate concentration depends upon the affinity of the enzyme for the substrate.

Question 55

What is the catalytic constant, k_cat ?

What is the equation for k_cat ?

Answer 55

k_cat is a turnover number.

It is the number of substrate molecules transformed per molecule of enzyme per second (units s^-1)

k_cat = v_max / [E₀]

Question 56

What is the equation for v₀ ?

Answer 56

v₀= (v_max . [S]) / ([S] + K_M )

Question 57

What is v_max?

Answer 57

On the Michaelis-Menten graph of v₀ against [S], the speed of the reaction increases with [S] but reaches a maximum point. This is the v_max.

The speed of the reaction cannot go any faster due to product inhibition; it will never reach full saturation.

Question 58

What is the specificity constant?

Answer 58

Enzymes can work on a number of different substrates. The efficiency of the on the substrate depends upon both k_cat and K_M.

specificity constant = k_cat / K_M

The higher the k_cat and the lower the K_M , the bigger the specificity constant.

The best substrate will have the highest specificity constant.

Question 59

What is competitive inhibition?

Answer 59

The inhibitor and substrate are very similar in size and shape. The substrate and inhibitor compete for binding at the active site.

This is reversible inhibition.

K_M is increased but v_max remains the same.

Question 60

What is uncompetitive inhibition?

Answer 60

There is no competition between the inhibitor and substrate for the active site.

The inhibitor binds to the enzyme-substrate complex, or once the E-S complex has been formed.

This type of inhibition is reversible.

K_M is unaltered (or appears reduced) but v_max is dramatically reduced.

Question 61

What is non-competitive inhibition?

Answer 61

This is a very rare type of inhibition.

The inhibitor can bind to both the free enzyme and the enzyme-substrate complex, but binds at sites distinct from the substrate.

This inhibition is irreversible.

v_max is reduced and K_M is not normally affected

Question 62

What is a lipid?

Answer 62

A molecule formed by linking a long-chain fatty acid to a glycerol-3-phosphate backbone; these are major components of biological membranes.

Question 63

What is the shape and properties of glycerophospholipids?

Answer 63

The charged polar head group and long chain non-polar tail mean the molecule is amphipathic.

Where more than one double bond occurs in the fatty acid chain, they are separated by 3 carbons.

Double bonds are nearly always cis ; this means they produce a pronounced kink in the shape of the acyl chain.

Tails with kinks in are more fluid, meaning that they can move around more in a membrane.

Question 64

What are the 3 types of membrane proteins?

Answer 64

Integral → sits in the membrane

Peripheral → associated with outer surface of the membrane

Lipid anchored → hydrophilic protein hanging into/out of cell