Enzymes (LOIL 3) Flashcards
state 2 introductory facts about - ‘Metabolic Regulation’
- many reactions are organised into pathways (dependent sequences of enzymatic reactions)
- pathways are regulated by feedback and feedforward control mechanisms
state the 2 main types of inhibition
- reversible inhibition
2. irreversible inhibition
state the 3 types of reversible inhibition
- competitive inhibition
- uncompetitive inhibition
- non-competitive inhibition
state the 3 types of irreversible inhibition
- group specific covalent modifying agents
- transition state analogues
- suicide inhibitors (mechanism-based inhibitors)
state 2 general facts about irreversible inhibition
- usually works by mimicking the substrate and resting with the active site (the method used by many pharmaceutical drugs)
- 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
explain how the following works - ‘group specific covalent modifying agents’
irreversible inhibitors that react with a specific functional group on an enzyme via covalent bonding
explain how the following works - ‘transition state analogues’
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
transition state inhibition can be useful for what 2 reasons ?
- understanding catalytic mechanisms (clues about the structure of the transition state)
- very specific inhibitors of enzymes (pharmaceutical applications)
explain what ‘suicide inhibitors’ are, and how they work (5 points)
- mechanism based inhibitors
- structurally similar to the substrate so ‘guides’ the reagent to the active site
- enzyme treats it as a substrate, starting chemical catalytic processes with the inhibitor
- chemical mechanism itself leads enzyme to react covalently with the inhibitor, thus ‘committing suicide’
- mechanism-based inhibition depends upon the chemical mechanism of catalysis
what is the main effect on enzymes/reactions in the presence of irreversible inhibitors
the target enzymes are inactivated until all of the irreversible inhibitor is used up
explain what ‘competitive inhibition’ is, and how it works (5 points)
- binds to the active site
- enzyme can bind to S or I, but not both
- proportion of enzyme that binds substrate and proportion that binds inhibitor is determined by relative concentrations
- inc^ [S] for a fixed [I] will inc^ the rate of reaction
- usually have a similar structure to the substrate
state the 2 things a Line-weaver Burk Plot will show about competitive inhibition
- km (apparent) increases
2. no effect on Vmax
1) what are NSAIDs ?
2) what do they do ?
- many Non-Steroidal Anti-Inflammatory Agents (NSAID’s)
2. competitive inhibitors of cyclooxygenase activity of PGH2 synthase
explain what ‘uncompetitive inhibition’ is, and how it works (4 points)
- substrate-dependent inhibition
- the inhibitor binds only to the ES complex
- binding site of an uncompetitive inhibitor is created on interaction of enzymes and substrates
- uncompetitive inhibition cannot be overcome by the addition of more substrates
state 2 things you can read off of a MM graph of ‘uncompetitive inhibition’
- because some unproductive ESI complex will always be present, Vmax will be lower I the presence of an inhibitor than in its absence
- km appears to have decreased because Vmax has decreased
state 2 things you can read off a Line-weaver Burk plot of ‘uncompetitive inhibition’
- km (apparent) decreases
2. Vmax decreases
explain what ‘non-competitive inhibition’ is, and how it works (4 points)
- changes the shape of the enzymes active site
- does this via an allosteric mechanism (other binding site)
- can bind to EITHER the free enzyme OR the ES complex
- decreases the concentration of functional enzyme
state 4 things you can read off of a MM graph about ‘non-competitive inhibition’
- reduced turnover rate (kcat)
- reduces Vmax
- km unchanged
- cannot be overcome by increasing the amount of substrate
why is km unchanged in ‘non-competitive inhibition’
km is unchanged as the inhibitor simply lowers functional [E], so the solution behaves more like a dilute solution of enzyme dose
state 2 things you can read off of a Line-weaver burk plot for ‘non-competitive inhibition’
- reduces Vmax
2. km unchanged
what are ‘Non Michaelis-Menten Enzymes’ ? (4 points)
- very sensitive systems
- simple inhibition is insufficient for metabolic needs (eg - resting —-> active)
- many regulated enzymes have two or more protein components - sub-units
- feedback in a multi-step reaction
state 5 facts about ‘Allosteric Enzymes’
- the alteration of one sub-unit can have an effect on the others
- do NOT obey MM kinetics
- may be cooperative in the binding of a substrate to one active site facilitates the binding of a substrate to another active site
- follow a ‘sigmoidal plot’
- often, an allosteric regulator is the end product of a pathway
what are ‘allosteric effectors’, and how do they work ? (4 points)
- binds to a site that alters the activity of the other sub-units (i.e. - it changes the protein conformation)
- does not bind to the active site (non-competitive)
- structurally unrelated to the substrate
- may activate or inhibit the enzymes action
state 5 things you can read off of a MM graph about ‘co-operative allosteric kinetics’
- small [I] or [A] causes a large ΔVo
- small curve shift, but huge effect on velocity
- much more sensitive than simple inhibition
- relatively small amount of effector needed
- relatively large effects
why is covalent regulation needed ?
sometimes, allosteric regulation is insufficient
covalent regulation can alter the activity of an enzyme dramatically by what 2 methods ?
- reversible (eg - phosphorylation)
2. irreversible (eg - irreversible)
state 2 method of reversible covalent regulation
- phosphorylation
2. adenylation (the addition of AMP to an enzyme)
explain, using 2 points, how phosphorylation works as a method of reversing covalent regulation
- addition of a phosphate group, usually to a serine or threonine residue on the enzyme
- not known how it works, but recent work suggests it’s a structural effect that moves an enzyme up to a higher energy state
state 3 facts about irreversible covalent regulation
- some enzymes are potentially very harmful (eg - proteases are quite happy to digest the host)
- enzymes synthesised in their inactive precursors are termed ‘zymogens’
- enzyme is irreversible cleaved from the zymogen (inactive form) to an active form via a protease
state what it is meant by the key term - ‘Zymogen’
a Zymogen the precursor of an enzyme that is produced in an inactive form
