Enzymes

Question 1

method of action of enzymes

Answer 1

• Enzymes can couple a spontaneous reaction to a nonspontaneous one, to make the overall ΔG < 0 (spontaneous)
• Reactions pass through high energy transition states.
• Activation energy is required to reach the transition state.
• Enzymes catalyse thermodynamically favourable reactions by lowering the activation energy
• The overall ΔG for the reaction is not changed.

Question 2

what are enzymes made of

Answer 2

usually proteins but occasionally RNA

Question 3

6 enzyme classes

Answer 3

1. oxidoreductase - used for redox
2. transferase - transfer of functional group
3. isomerases - transfer of atoms/groups withina molecule to form isomer
4. lyases - non-hydrolytic breaking or making of bonds
5. ligases - join two molecules together
6. hydrolase - hydrolysis reactions

Question 4

cofactors

Answer 4

non-protein factors which help catalyse reactions. Can be metal ions or coenzymes

Question 5

metal ions as cofactors

Answer 5

• Are Lewis acids (i.e. election acceptors), so they can participate in acid-base catalysis
• Form coordination compounds with precise geometries (good for positioning reactants exactly where they need to be).
• e.g. Mg2+ used for DNA polymerase

Question 6

coenzymes

Answer 6

• Small organic molecules.
• Co-substrates - required for enzyme-substrate complex interaction, formation or stabilisation
• Carriers (of electrons, atoms or functional groups)
• Often derived from vitamins

Question 7

features of active site

Answer 7

• has amino acid side chains pointing into it
• binds substrate via several initial weak interactions
• determines specificity
• initial weak bonds are remodelled to form transition state

Question 8

types of ES bonds

Answer 8

• ionic bonds - charged side chains
• hydrogen bonds - O and N atoms in side chains or backbones
• VDW’s interactions - between any protein and substrate in close proximity, weakest
• covalent bonds - rare, v strong

Question 9

why are weak bonds advantageous

Answer 9

• easy to break when complex breaks apart - reversibility
• Weak bonds can only form if the relevant atoms are precisely positioned - specificity

Question 10

what does stereospecificity mean?

Answer 10

enzymes can recognise between different enantiomers (chiral compounds)

Question 11

lock and key model

Answer 11

Substrate and active site have exactly complementary shapes

Question 12

induced fit model

Answer 12

• Active site conformation changes slightly when substrate tries to bind
• Shows that enzymes are dynamic, not static

Question 13

3 ways ΔGe‡ is lowered

Answer 13

1. Ground state destabilisation - free energy increases
2. Transition state stabilisation - free energy decreases
3. Alternate reaction pathway with a different (lower energy) transition state

(1) and (2) can be achieved the same way: by having an active site that has shape/charge complementarity to the TS, not the substrate

Question 14

should an enzyme bind to substrate or transition state more tightly?

Answer 14

transition state however this is difficult because it is transient and cannot be isolated

Question 15

5 catalytic mechanisms

Answer 15

1. preferential binding of transition state
2. proximity and orientation effects - need to be close together and right orientation to react
3. acid base catalysis - His is particularly suitable because has pKa 6.5, close to body pH so can donate or accept a proton depending on environment of active site
4. metal ion catalysis - provide substrate orientation, ability to act as Lewis acids, sites for electron transfer
5. covalent catalysis - formation of a reactive, short-lived intermediate, which is covalently attached to the enzyme

Question 16

progress curve

Answer 16

• measures the appearance of product (or disappearance of substrate) with time
• Important to measure initial reaction velocity (rate) i.e. at time zero
• passes through origin

Question 17

factors affecting reaction rate

Answer 17

• temperature - increases until optimum
• pH - optimum
• amount of enzyme is increased, the rate of reaction increases, provided substrate is in excess
• As amount of substrate increases, rate of reaction increases linearly until all active sites are occupied, at which point rate stops increasing

Question 18

Vmax

Answer 18

• maximum velocity possible when [S] = infinity
• on V vs [S] curve, this is horizontal asymptote
• on Lineweaver-Burk plot, this is 1/y-int

Question 19

what is Km

Answer 19

• Michaelis constant
• substrate conc at which V = Vmax/2
• on Lineweaver-Burk plot, Km = -1/x-int

Question 20

significance of Km

Answer 20

• Substrate conc needed to reach half Vmax
• Low KM = high affinity between E and S
• High KM = low affinity
• In the cell, for a particular enzyme-substrate interaction, [S] is often below the Km, allowing for rate control

Question 21

k_cat

Answer 21

• number of substrate molecules converted to product, per enzyme, per unit of time, when E is saturated with substrate
• If Michaelis-Menten model fits then Vmax = kcat[E_total]
• high kcat is good

Question 22

michaelis-menten equation and assumptions

Answer 22

assumptions:

• Product is not converted back to substrate.
• the rate of ES formation equals the rate of its breakdown hence no change in [ES]
• Measuring initial rate means [S] does not change significantly (and [S] is greater than [E])

Question 23

k_cat/Km

Answer 23

overall measure of enzyme efficiency

Question 24

irreversible inhibitor

Answer 24

• Binds covalently to enzyme, permanently inactivating it
• Inhibitor reacts with a specific amino acid side chain, usually in the active site, and forms a covalent bond
• e.g. natural toxins

Question 25

reversible inhibitor

Answer 25

• not covalently bound to enzyme
• can be competitive or non-competitive

Question 26

competitive inhibition

Answer 26

• Depends on relative concentrations of substrate and inhibitor
• Competes directly with substrate for active site
• No change in Vmax - Infinite [S] outcompetes the inhibitor
• Increases KM - More substrate is needed to get to V = Vmax / 2

Question 27

non-competitive inhibitor

Answer 27

• Inhibitor binds does not bind to active site
• Enzyme can bind substrate, or inhibitor, or both
• can be pure non-CI or mixed non-CI

Question 28

pure non-CI inhibitor

Answer 28

• Binding of Inhibitor has no effect on the binding of S; i.e. the substrate binds to E and EI with the same affinity
• Binding Inhibitor changes the structure of the active site such that S still binds, but transition state stabilisation is no longer optimal.
• Vmax decreases; KM stays the same

Question 29

mixed non-CI inhibitor

Answer 29

• More commonly, binding of the inhibitor does affect binding of the substrate
• Vmax decreases; KM increases

Question 30

methods of enzyme regulation

Answer 30

1. Turn gene expression on or off
2. Degrade enzyme
3. Covalent modification e.g. phosphorylation
4. Proteolytic cleavage
5. Allosteric effects in multisubunit enzymes

Question 31

regulation by proteolysis

Answer 31

• Some enzymes are synthesized as zymogens.
• The full length polypeptide is inactive
• Proteases cleave the zymogen polypeptide and remove a peptide or peptides to activate the enzyme
• Many digestive proteases (e.g. trypsin, chymotrypsin) and blood clotting proteins are made as zymogens

Question 32

feedback inhibition

Answer 32

• turning an enzyme on AND off
• A sensible strategy is to avoid making unnecessary metabolic intermediates
• Final product blocks an early reaction in a series and shuts down whole series

Question 33

allosteric effect

Answer 33

• V vs. [S] plot is a sigmoid, not a hyperbola (like Hb O2 binding curve) (shows Michaelis-Menten curve)
• Allosteric enzymes have multiple subunits
• They display cooperative behaviour:
• Binding of one substrate to the first subunit makes it easier for the second substrate to bind, which makes it easier for the third substrate to bind
• allosteric activators and inhibitors bind to site other than active site
• activator shifts curve left
• inhibitor shifts curve right

Question 34

lineweaver-burke plot

Answer 34

• reciprocal of michaelis-menten equation
• x-int = -1/Km
• y-int = 1/Vmax
• slope = Km/Vmax

Question 35

effect of heat on a protein

Answer 35

• irreversibly break non covalent bonds (H-bonds) and reform them with other proteins (primarily hydrophobic) – see precipitation with it being unable to dissolve.
• Breaks all the way down to 2o structure

Question 36

effect of salt on protein

Answer 36

• removes water from around it, freeing up interactions to other proteins – no real change to 2o structure, and minimal to tertiary (H-bonds mostly the same also).
• Become more compact and shrink so is More stable.
• Can be redissolved

Question 37

what is tyrosinase and what can bind to its active site

Answer 37

it is a copper-containing oxidoreductase. Tyrosine, dopamine and O2 can bind to its active site

Question 38

regulation of enzymes by proteolysis

Answer 38

• some enzymes synthesised as zymogens (inactive form)
• proteases cleave zymogen to activate enzymes
• allows for temporal and spacial control