Enzymes (LOIL 3)

Question 1

state 2 introductory facts about - ‘Metabolic Regulation’

Answer 1

1. many reactions are organised into pathways (dependent sequences of enzymatic reactions)
2. pathways are regulated by feedback and feedforward control mechanisms

Question 2

state the 2 main types of inhibition

Answer 2

1. reversible inhibition

2. irreversible inhibition

Question 3

state the 3 types of reversible inhibition

Answer 3

1. competitive inhibition
2. uncompetitive inhibition
3. non-competitive inhibition

Question 4

state the 3 types of irreversible inhibition

Answer 4

1. group specific covalent modifying agents
2. transition state analogues
3. suicide inhibitors (mechanism-based inhibitors)

Question 5

state 2 general facts about irreversible inhibition

Answer 5

1. usually works by mimicking the substrate and resting with the active site (the method used by many pharmaceutical drugs)
2. an irreversible inhibitor usually dissociates very slowly from its target enzyme as it has become tightly bound to the enzyme; either covalently or non-covalently

Question 6

explain how the following works - ‘group specific covalent modifying agents’

Answer 6

irreversible inhibitors that react with a specific functional group on an enzyme via covalent bonding

Question 7

explain how the following works - ‘transition state analogues’

Answer 7

structurally similar to the transition state, but bind more tightly to the enzyme than the substrate does; has a very high affinity for the active site

Question 8

transition state inhibition can be useful for what 2 reasons ?

Answer 8

1. understanding catalytic mechanisms (clues about the structure of the transition state)
2. very specific inhibitors of enzymes (pharmaceutical applications)

Question 9

explain what ‘suicide inhibitors’ are, and how they work (5 points)

Answer 9

1. mechanism based inhibitors
2. structurally similar to the substrate so ‘guides’ the reagent to the active site
3. enzyme treats it as a substrate, starting chemical catalytic processes with the inhibitor
4. chemical mechanism itself leads enzyme to react covalently with the inhibitor, thus ‘committing suicide’
5. mechanism-based inhibition depends upon the chemical mechanism of catalysis

Question 10

what is the main effect on enzymes/reactions in the presence of irreversible inhibitors

Answer 10

the target enzymes are inactivated until all of the irreversible inhibitor is used up

Question 11

explain what ‘competitive inhibition’ is, and how it works (5 points)

Answer 11

1. binds to the active site
2. enzyme can bind to S or I, but not both
3. proportion of enzyme that binds substrate and proportion that binds inhibitor is determined by relative concentrations
4. inc^ [S] for a fixed [I] will inc^ the rate of reaction
5. usually have a similar structure to the substrate

Question 12

state the 2 things a Line-weaver Burk Plot will show about competitive inhibition

Answer 12

1. km (apparent) increases

2. no effect on Vmax

Question 13

1) what are NSAIDs ?

2) what do they do ?

Answer 13

1. many Non-Steroidal Anti-Inflammatory Agents (NSAID’s)

2. competitive inhibitors of cyclooxygenase activity of PGH2 synthase

Question 14

explain what ‘uncompetitive inhibition’ is, and how it works (4 points)

Answer 14

1. substrate-dependent inhibition
2. the inhibitor binds only to the ES complex
3. binding site of an uncompetitive inhibitor is created on interaction of enzymes and substrates
4. uncompetitive inhibition cannot be overcome by the addition of more substrates

Question 15

state 2 things you can read off of a MM graph of ‘uncompetitive inhibition’

Answer 15

1. because some unproductive ESI complex will always be present, Vmax will be lower I the presence of an inhibitor than in its absence
2. km appears to have decreased because Vmax has decreased

Question 16

state 2 things you can read off a Line-weaver Burk plot of ‘uncompetitive inhibition’

Answer 16

1. km (apparent) decreases

2. Vmax decreases

Question 17

explain what ‘non-competitive inhibition’ is, and how it works (4 points)

Answer 17

1. changes the shape of the enzymes active site
2. does this via an allosteric mechanism (other binding site)
3. can bind to EITHER the free enzyme OR the ES complex
4. decreases the concentration of functional enzyme

Question 18

state 4 things you can read off of a MM graph about ‘non-competitive inhibition’

Answer 18

1. reduced turnover rate (kcat)
2. reduces Vmax
3. km unchanged
4. cannot be overcome by increasing the amount of substrate

Question 19

why is km unchanged in ‘non-competitive inhibition’

Answer 19

km is unchanged as the inhibitor simply lowers functional [E], so the solution behaves more like a dilute solution of enzyme dose

Question 20

state 2 things you can read off of a Line-weaver burk plot for ‘non-competitive inhibition’

Answer 20

1. reduces Vmax

2. km unchanged

Question 21

what are ‘Non Michaelis-Menten Enzymes’ ? (4 points)

Answer 21

1. very sensitive systems
2. simple inhibition is insufficient for metabolic needs (eg - resting —-> active)
3. many regulated enzymes have two or more protein components - sub-units
4. feedback in a multi-step reaction

Question 22

state 5 facts about ‘Allosteric Enzymes’

Answer 22

1. the alteration of one sub-unit can have an effect on the others
2. do NOT obey MM kinetics
3. may be cooperative in the binding of a substrate to one active site facilitates the binding of a substrate to another active site
4. follow a ‘sigmoidal plot’
5. often, an allosteric regulator is the end product of a pathway

Question 23

what are ‘allosteric effectors’, and how do they work ? (4 points)

Answer 23

1. binds to a site that alters the activity of the other sub-units (i.e. - it changes the protein conformation)
2. does not bind to the active site (non-competitive)
3. structurally unrelated to the substrate
4. may activate or inhibit the enzymes action

Question 24

state 5 things you can read off of a MM graph about ‘co-operative allosteric kinetics’

Answer 24

1. small [I] or [A] causes a large ΔVo
2. small curve shift, but huge effect on velocity
3. much more sensitive than simple inhibition
4. relatively small amount of effector needed
5. relatively large effects

Question 25

why is covalent regulation needed ?

Answer 25

sometimes, allosteric regulation is insufficient

Question 26

covalent regulation can alter the activity of an enzyme dramatically by what 2 methods ?

Answer 26

1. reversible (eg - phosphorylation)

2. irreversible (eg - irreversible)

Question 27

state 2 method of reversible covalent regulation

Answer 27

1. phosphorylation

2. adenylation (the addition of AMP to an enzyme)

Question 28

explain, using 2 points, how phosphorylation works as a method of reversing covalent regulation

Answer 28

1. addition of a phosphate group, usually to a serine or threonine residue on the enzyme
2. not known how it works, but recent work suggests it’s a structural effect that moves an enzyme up to a higher energy state

Question 29

state 3 facts about irreversible covalent regulation

Answer 29

1. some enzymes are potentially very harmful (eg - proteases are quite happy to digest the host)
2. enzymes synthesised in their inactive precursors are termed ‘zymogens’
3. enzyme is irreversible cleaved from the zymogen (inactive form) to an active form via a protease

Question 30

state what it is meant by the key term - ‘Zymogen’

Answer 30

a Zymogen the precursor of an enzyme that is produced in an inactive form