Enzymes Flashcards
What is a cofactor?
A non-protein factor that helps and enzyme catalyse a reaction
What are the 2 classes of cofactor?
- metal ion
- coenzyme
Are metal ion cofactors Lewis acids or bases?
Lewis acids = e- acceptors
What is the function of metal ion cofactors?
form coordination compounds with precise geometries - good for positioning reactants precisely where they need to be in the active site
What are coenzymes?
- small organic molecules
- co-substrates (involved at different steps, but not used up in the reaction)
- carriers (of e-, atoms or functional groups)
- often derived from vitamins
Describe the cofactor used in glycogen phosphorylation
PLP cofactor at active site of glycogen phosphorylate and facilitates its activity
What are advantages of using many weak bonds to bind the S to E?
○ Strength in numbers
○ Several bonds required for substrate and specificity
○ Only form if the relevant atoms are precisely positioned
○ Easy to break = reversibility
How is Ea lowered? (3)
- Ground state destabilisation
- Ground state energy up
- Transition state stabilisation
- Alternate reaction pathway with different (lower energy) transition state
How can gs destabilisation and ts stabilisation be achieved?
by having an active site that has shape/charge complementarity to the TS, not the substrate
Name the 5 catalytic mechanisms
- Preferential binding of the transition state.
- Proximity and orientation effects.
- Acid-base catalysis
- Metal ion catalysis.
- Covalent catalysis.
What is an analogue?
Resembles the transition state but doesn’t disappear, is more stable
- Very effective as drugs
What is an example of an analogue? What is its clinical function?
Lipitor = powerful cholesterol lowering drug
How does lipitor work?
Inhibits HMG-CoA reductase
- Natural substrate = has aldehyde group
- TS analogue = alcohol group -> binds better to Lipitor because it resembles the transition state, rather than the normal substrate
Lipitor can be classified as a…
transition state analogue inhibitor of the reaction
Proximity and orientation effects refers to…
…that for 2 molecules to react, they need to be in close proximity and at correct orientation
Acid-base catalysis involves…
proton (H+) transfer
Which amino acids will donate a proton, and at what pH?
- Glu, Asp give up proton at low pH = -ve charge
- Lys, Arg will only donate at high pH
What is special about histidine in terms of acid-base catalysis?
Histidine can either accept or donate a proton depending on environment
- pKa(His) ~ 6.5 (close to physiological pH) -> can act as acid or base
In metal ion catalysis, the metal ion provides…
- Substrate orientation (due to specific coordination geometries)
- Ability to act as Lewis acids (e- acceptors) to polarize water/other functional groups
- Provide sites for e- transfer (for catalyzing Redox reactions)
What is an example of an enzyme which uses metal ion catalysis?
Hexokinase
- Large -ve charge on O’s in TS
- Mg2+ balances the -ve charge of the transition state
.: Decreases energy of transitions state
Describe what is formed in covalent catalysis
A reactive, short-lived intermediate is formed, covalently attached to the enzyme
Define enzyme inhibitor
a compound that binds to an enzyme and reduces its activity
What are the 2 classes of inhibitors?
irreversible and reversible
how/where does an irreversible inhibitor bind?
binds covalently to the enzyme by reacting with a specific aa side chain in the active site
how/where does a reversible inhibitor bind?
not covalently bound to the enzyme, can be competitive with the substrate (trying to bind to same place), non-competitive, or a combo of both (= mixed inhibitor)
what is the difference between a reversible and irreversible inhibitor?
irreversible: binds to enzyme and permanently inactivates it [covalent bond]
reversible: binds to the enzyme but can be released (leaves the enzyme in its original condition)
what is a competitive inhibitor?
Inhibitor competes directly with the substrate for the active site
can a competitive inhibitor be outcompeted by the substrate?
yes, infinite [S] outcompetes the inhibitor
What changes to kinetic parameters occur when a competitive inhibitor is added?
More substrate needed to get to V = Vmax/2 → No change in V max - No change in y intercept → Increase in Km - X intercept moves closer to 0
What is an example of a competitive inhibitor? (2)
• Transition state analogues (e.g. Lipitor)
• Anastrozole - inhibits aromatase
→ Treatment of breast cancer
true or false: in non-competitive inhibition, the enzyme can bind substrate or inhibitor, not both
false: enzyme can bind substrate or inhibitor or both because inhibitor binds at different site than substrate
What is pure non-competitive inhibition?
where the binding of I has no effect on the binding of S
What changes to kinetic parameters occur when a pure non-competitive inhibitor is added?
→ Vmax decreases
→ Km stays the same, because ability of substrate binding to active site is not affected by the enzyme
What is mixed inhibition?
When inhibitor binding slightly changes substrate affinity
What changes to kinetic parameters occur when a mixed inhibitor is added?
→ Vmax and Km both change
What is the function of glycogen phosphorylase?
Takes the terminal sugar off the glycogen (which is stored in muscles) , then used to derive energy
How is glycogen phosphorylase activity regulated?
by both allosteric activators (AMP) and inhibitors (feedback inhibition of glycogen phosphorylase)
A sigmoidal curve shows what about Michaelis-Menten kinetics?
That allosteric enzymes break Michaelis-Menten parameters
