Lecture 13. Enzymes and Catalysis Flashcards
What does Eyring’s equation show ?
The rate of reaction can be related to Gibbs free energy of the transition state for that reaction
What is the activation state ?
The energy required to get to the transition state from starting materials
What happens as the activation energy increases ?
The rate constant (k) becomes smaller
The reaction becomes slower
What can catalysts do ?
- Reduce the activation energy for a reaction
2. Provide an alternative mechanism with a lower overall activation energy
Why are enzymes crucial for life ?
Activation energies for uncatalysed metabolic processes are so big that very high temperatures would be requires to achieve the same rates
What does a large difference in the activation energy result in ?
A larger difference in k catalysed and k uncatalysed
What can enzymes provide ?
A large reduction in the activation energy
What are the four categories by which enzymes catalyse reactions ?
- Proximity and orientation effects
- Acid base catalysis
- Covalent catalysis
- Transition state stabilisation
What type of cost does binding a substrate to an enzyme require ?
Entropic cost
What is required for a substrate to bind tightly to an enzyme ?
Favourable enthalpy
What does binding of substrate molecules to enzymes allow ?
The enzyme to exert control over the conformation and shape of the substrate molecule
What does free rotation around single bonds in acyclic systems result in ?
A huge number of thermally accessible conformations
Which type of reaction (intramolecular or intermolecular) are generally faster ?
Intramolecular
Why are intramolecular reactions generally faster ?
The reactants are already in the molecule
What can contribute to influencing the rate of acceleration ?
- Different functionality from different amino groups
- Shape and form of protein
- Interactions with the amide backbone
In bimolecular reaction what are proximity and orientation effects thought to account for ?
Accelerations up to 10^8 fold over the background uncatalysed reaction
How can protonation affect functional groups in substrate molecules ?
Turn them into better leaving groups
How does protonation affect carbonyl groups ?
Makes them more electrophilic
What can deprotonation do to functional groups ?
Make them into better nucleophiles
What does acid/base catalysis do by appropriate amino acid side chains ?
Accelerating enzymatic processes
What amino acid cannot be chiral ?
Glycine
What amino acids have carboxylic side chains ?
Aspartic acid and glutamic acid
What happens if aspartic acid and glutamic acid are protonated in the active sites ?
They can act as proton donors
What happens if aspartic acid and glutamic acid are deprotonated ?
They can stabilise adjacent positive charges and perhaps act as bases (proton acceptors)
What happens when the pH of an environment corresponds to the molecules pKa ?
The compound will be 50% ionised
What can nucleophilic sides chains react directly with and form ?
Substrates forming covalent bonds between the enzyme and the substrate
What happens to the covalent bonds formed between the enzyme and substrate ?
Cleaved in later steps, regenerating the unmodified enzyme
What can lysine react with ?
Carbonyl groups in substrates to form imines/iminium ions
What does thioester formation occur with ?
Cysteine side chains
What can favorable reactions between the enzyme and substrate do ?
Stabilise the transition state and lower the activation energy
What are some methods of understanding how an enzyme works ?
- Kinetic studies
- Detection of intermediates
- Chemical modification
What is x-ray crystallography used for?
A very powerful technique that allows for the 3D structure of the enzyme to be solved
How would you obtain the structure of the enzyme-substrate complex ?
Grow crystals and soak in substrates
What is the problem of growing and soaking the crystals in substrate ?
Enzyme turnover is generally much faster than the timescale of crystallisation and data aquisition
How would you solve the problem of a quick enzyme turnover in x-ray crystallography ?
- Use an unreactive substate mimic
- If the enzyme requires two substrates, try two crystals one with each substrate
- Complex of the enzyme with the product
What can the use of x-ray crystallography in conjunction with computational modelling do ?
Reverse some of the tricks that are used to obtain crystal structures
What is site directed mutagenesis ?
Amino acids are exchanged for others
Which amino acids should you mutate ?
Target key structural/catalytical residues
In site directed mutagenesis, how would you make as little disruption as possible to the enzyme structure ?
- Remove the reactive group
2. Replace with something of similar size (ideally smaller) and polarity/charge distribution
