Lecture 10 - Enzyme Catalyse a reaction Flashcards
Strategies for Catalysis (6)
- Acid-base catalysis
- Covalent catalysis
- Redox and radical catalysis (metal ions)
- Geometric effects (proximity and orientation)
- Stabilisation of the transition state
- Cofactors with activated groups, e.g. electrons, hydride ion (H-), methyl groups (CH3), amino groups (NH2).
Do many enzymes use more than one from the strategies for catalysis list?
Yes
not exclusive
not exhaustive
For two molecules to react they need to be:
Close together
right orientation
Covalent catalysis
Involves formation of a reactive, short-lived intermediate, which is covalently attached to the enzyme.
what drives covalent catalysis?
Nucleophilic attack on an electrophile
Good electrophiles
Positive
Centres that able to form additional bonds
Good electrophiles examples
Carbonyl carbon atom Cationic imine (Schiff base)
Good nucleophiles
Things that attack electrophiles
Good nucleophiles examples
Hydroxyl group (need to be deprotonated) Sulfhydryl group (need to be deprotonated) Amino group (uncharged state) Imidazole group (uncharged state) Found in enzyme active sites
what does a nucleophilic attack require?
correct orientation
Ionisation
acid-base catalysis
Involves ionisable groups and proton transfer.
what may be part of the activation to the transition state?
Ionisation
why do Groups need to be in correct ionisation state?
For catalytic mechanism to proceed.
Ionisable group that can easily lose a proton
Glu
Asp
Ionisable group that can easily gain a proton
Lys
Arg
Ionisable groups that most active in acid base catalysis and assist with forming better nucleophiles
Glu Asp
Lys Arg
Enzyme activity is
pH dependent.
what do each enzyme have at which its rate is highest?
characteristic optimal pH
what do Amino acid sidechains need to be in?
correct ionisation state for catalytic mechanism to proceed.
what example is suitable to acid base catalysis, types of reactions?
Histidine
Histidine
Imidazole pKa
~ 6.5 – close to physiological pH
what can the active site His depending on environment do?
Donate or accept proton
Divergent evolution of serine proteases
incl examples
Same structure, unique specificities (pocket)
Chymotrypsin, Trypsin, Elastase
Convergent evolution of serine proteases
Same catalytic triad occurs in different order and in different structures.
Catalytic triad
Asp, his, ser
Convergent evolution
Occur in 3 different order and structure in 3 different proteins
In Chymotrypsin: acid - base and covalent catalysis.
what does the Catalytic triad comprise of?
serine, histidine and aspartic acid.
In Chymotrypsin: acid - base and covalent catalysis.
How does Serine Hydroxyl become a good nucleophile?
Sharing proton with histidine
In Chymotrypsin: acid - base and covalent catalysis.
What does Serine Hydroxyl attack?
Scissile bond
In Chymotrypsin: acid - base and covalent catalysis.
What does the Oxyanion hole do?
Stabilise tetrahedral intermediate
In Chymotrypsin: acid - base and covalent catalysis.
what is polarised by Asp102?
His57
In Chymotrypsin: acid - base and covalent catalysis.
what does His57 withdraw proton from?
Ser195
becomes nucleophilic
In Chymotrypsin: acid - base and covalent catalysis.
what does Non-polar environment of Asp102 do?
raises its pKa.
Oxyanion hole stabilise tetrahedral intermediate.
In Chymotrypsin: acid - base and covalent catalysis.
What drives decomposing of tetrahedral intermediate?
acid catalysis from His57.
In Chymotrypsin: acid - base and covalent catalysis.
what happens to both halves of polypeptide ‘acyl-enzyme intermediate’?
Half polypeptide remains covalently attached to the enzyme.
other half can leave the active site.
In Chymotrypsin: acid - base and covalent catalysis.
what replaces one of the products in the active site in Acyl-enzyme intermediate?
Water
In Chymotrypsin: acid - base and covalent catalysis.
what is Polarised by His57 as a general base?
water
In Chymotrypsin: acid - base and covalent catalysis.
what does water make?
nucleophilic attack
In Chymotrypsin: acid - base and covalent catalysis.
when water makes a nucleophilic attack what does it form?
incl how its stabilised
second tetrahedral intermediate
Stabilised by oxyanion hole
In Chymotrypsin: acid - base and covalent catalysis.
what acts as acid when 2nd intermediate decays?
His57
In Chymotrypsin: acid - base and covalent catalysis.
what does 2nd intermediate form when it decays?
Carboxylate
In Chymotrypsin: acid - base and covalent catalysis.
what happens when 2nd intermediate decays?
2nd product leave the active site.
Active site has been regenerated and is ready for
another round of catalysis.
Aspartic protease mechanism examples
HIV protease and pepsin
what is versatile?
Acid-base catalysis
Catalytic triad occurs by
convergent and
divergent evolution.
Stabilisation of electrophilic targets invites
nucleophilic attack.
Stabilisation of an intermediate is
crucial to many reactions.
Covalent catalysis can be resolved by
complementary half reaction.
