Lecture 10: HOW DOES AN ENZYME CATALYSE A REACTION? 1 Flashcards
What are the strategies for catalysis?
Acid-base catalysis, covalent catalysis, redox and radical catalysis, geometric effects, stabilisation of the transition state and cofactors with activated groups
What is redox and radical catalysis driven by?
Metal ions
What are the geometric effects?
Proximity and orientation
what are some examples of activated groups of cofactors?
Electrons, hydride ions (H-), methyl groups (CH3), amino groups (NH3) with the ability to carry things
What do many enzymes do?
Use more than one strategy
What does covalent catalysis involve?
The formation of a reactive, short-lived intermediate, which is covalently attached to the enzyme
What does the enzyme in covalent catalysis have?
A nucleophilic side chain
How is the enzyme regenerated in covalent catalysis?
By a hydrolysis reaction
What is covalent catalysis driven by?
Nucleophilic attack on an electrophile
For two molecules to react they need to be …
Close together and in the correct orientation
What does nucleophilic attack require?
Correct orientation and ionisation
What does nucleophilic attack depend on?
The R groups and how good the leaving group is
What does nucleophilic attack form?
A trigonal planar intermediate
What should allow nucleophilic attack to occur faster?
Stabilising the transition state
What does hexokinase use as a cofactor?
Mg2+
What is established in hexokinase?
Orientation of phosphates of ATP by octahedral coordination of Mg2+ ion
What is the Mg2+ cofactor known as?
An electron withdrawing Lewis acid which stabilises oxygen, making phosphorus a better electrophile (leaving group)
What does acid-base catalysis involve?
Ionisable groups and proton transfer
What amino acids have ionisable groups?
Glu, Asp (COOH)
Lys, Arg (NH3+)
What do amino acid groups need to be in for the catalytic mechanism to proceed?
In the correct ionisation state
What may ionisation be part of?
activation to the transition state
What is enzyme activity dependent on?
pH
Why is enzyme activity ph dependent?
Because transfer of protons is involved
What does each enzyme have?
A characteristic optimal pH at which its rate is highest
What is histidine particularly suitable to?
Acid-base catalysis reactions
What is the pKa of the imidazole group in histidine?
~6.5 which is close to physiological pH
What can histidine do?
Depending on the environment of the active site His can donate or accept a proton
What does chymotrypsin do?
Cleave proteins
What catalysis strategies does chymotrypsin use?
Acid-base, covalent, geometric effects and stabilisation of the transition state
What are proteases?
Hydrolyases
What are the substrates in a reaction using chymotrypsin?
A polypeptide and water
What are the products in a reaction using chymotrypsin?
Two shorter peptides, or individual amino acids
What does chymotrypsin act in?
Digestion
What do other proteases do?
Make hormones, remove defunct proteins we no longer need, or activate enzymes (as in blood clotting)
What do divergent evolution serine proteases have?
Same structure with unique specificities
What is in the specificity pocket of chymotrypsin?
2 glycines (small)
What is in the specificity pocket of trypsin?
Negative aspartic acid which attracts positively charged polypeptides
What is in the specificity pocket of elastase?
Valine and threonine with large side chains
What do convergent serine proteases have?
The same catalytic triad occurs in different order and different structures.
What should proteases have if they shared a common ancestor?
Share order and structure
What does the catalytic triad in chymotrypsin include?
Serine, histidine and aspartic acid
What is the first part in the action of chymotrypsin?
his57, polarised by Asp102, withdraws a proton from Ser195 making there serine hydroxyl a good nucleophile to attack the sissile bond
What is the sissile bond?
The bond which is broken
What happens after his57, polarised by Asp102, withdraws a proton from Ser195 making there serine hydroxyl a good nucleophile to attack the sissile bond?
Nucleophilic attack forms a covalent, tetrahedral intermediate which is stabilised by the oxyanion hole, lowering the activation energy
what happens after Nucleophilic attack forms a covalent, tetrahedral intermediate which is stabilised by the oxyanion whole, lowering the activation energy?
The tetrahedral intermediate decomposes, driven by general acid catalysis from his57
What happens as a result of the first tetrahedral intermediate decomposing?
Half of the polypeptide chain remains covalently attached to the enzyme in an acyl-enzyme intermediate and the other half can leave the active site
What happens after the first half leaves the active site?
Water replaces one of the products in the active site
What happens after water replaces one of the products in the active site?
Polarised by His57 acting as a general base (OH-), water makes a nucleophilic attack forming a second tetrahedral intermediate, again stabilised by the oxyanion hole
What happens after Polarised by His57 acting as a general base (OH-), water makes a nucleophilic attack forming a second tetrahedral intermediate, again stabilised by the oxyanion hole?
Whit His57 acting as a general acid, this intermediate decays to form a carboxylate
What happens as a result of the second intermediate decaying to form a carboxylate?
The second product can leave the active site and the active site has been regenerated and is ready for another round of catalysis
