Chemical Catalysis

Question 1

How do proximity and orientation effect the Gibbs free energy?

Answer 1

When both are tethered to the same molecule, for example the enzyme, the rate changes to a first order reaction. When the enzyme is used there is a decrease in the rotational and translational entropy.

Question 2

How does electrostatic catalysis effect the Gibbs Free Energy?

Answer 2

The binding Of a substrate to the active site excludes water. This greatly changes the dielectric constant of the medium resulting in strong electrostatic interactions. This can have a significant effect on the Pka of the active site. Charge distributions in active sites are thought to be arranged so as to stabilise transition states of enzyme catalysed reactions.

Question 3

How do general acids or general bases stabilise transition states?

Answer 3

General bases abstract protons, stabilising the developing positive charge by transfer of a proton to the base. When there are Unsolved charges there is high energy which can make the structure destabilised.
General acids donate protons, stabilising the developing negative charge by donating a proton. Unresolved charges induce instability.

Question 4

Describe the reaction rate in specific acid base catalysis.

Describe the reaction rate on general acid base catalysis.

Answer 4

Specific acid base catalysis: the reaction rate is directly proportional to the concentration of protons and hydroxyl ions.
General acid base catalysis: the reaction rate is proportional to the concentration of the Brønsted acid or Brønsted base.

Question 5

Is general acid base catalysis a fast or slow process, why?

Answer 5

It is a fast process because it utilises both acid and base catalysis to stabilise the transition state.

Question 6

Give an example of a classic general acid base catalysis mechanism.

Answer 6

Bovine pancreatic RNase A.

It digests oligonucelotides and has a bell shaped curve pH activity profile.

Question 7

Describe some evidence for the RNase mechanism.

Answer 7

The bell shaped activity Curve for RNase is between 5.4 and 6.4 which means the catalytic role of the functional groups is between 5.4 and 6.4, likely to be histidines.
The crystal structure of the RNase complex with cytidine 2,3 cyclic phosphate intermediate. Hydrogen bonding interactions between cytosine and Threonine 45 that confer substrate specificity.
Chemical modification, alkylation of either histidine halts catalysis, binding of substrates protects histidines from modification.

Question 8

How can chemical rates be accelerated by covalent catalysis?

Answer 8

Enzymes can accelerate rates by transient formation of covalent bonds between the substrate and enzyme. It is usually a nucleophilic attack by the enzyme on the electrophilic centre of a substrate.

Question 9

Name some important nucleophile groups and some important electrophile groups.

Answer 9

Nucleophile groups: imidazole,
Amino,
Hydroxyl
Electrophile groups: protons,
Metal ions,
Carbonyl carbons

Question 10

What is a schiff base?

How does it stabilise the transient state?

Answer 10

Formation of a schiff base is an example of the first step of covalent catalysis, linking the catalyst to a substrate through nucleophilic addition. The protonated nitrogen atom of a schiff base can serve as an electron sink.
The schiff base serves as an electron sink to reduce the otherwise high energy enolate character of the transient state.

Question 11

How do metal ions participate in catalysis?

Answer 11

Binding substrates and properly orienting them for reactions,
Mediating oxidation- reduction reactions through reversible changes in the metal oxidation state.
Electrostatically stabilising or shielding negative charges,
Providing a source of hydroxyl ions at a neutral pH.

Question 12

Protein crystallography can be used to produce high resolution structures of what?

Answer 12

Native and mutant enzymes,

Enzyme ligand complexes- such as inhibitors and substrates.

Question 13

What are the steps involved in solving protein structures using crystallography?

Answer 13

Protein expression,
Crystallisation,
Diffraction data collection,
Phasing,
Map interpretation,
Refinement,
Analysis.

Question 14

Why is a crystal structure used?

Answer 14

Because the X-ray scattering from a single molecule is weak. A crystal amplifies the X-ray scattering.

Question 15

What is supersaturation?

Answer 15

This is achieved by adding more of a substance to a solution than can normally be dissolved. This is a thermodynamically unstable state.

Question 16

What is the most commonly used method of protein crystallisation?

Answer 16

Vapour diffusion.

Question 17

Describe vapour diffusion.

Answer 17

In this method a droplet containing purified protein, precipitant and buffer solutions is allowed to equilibrate with a larger reservoir of more concentrated precipitants and buffer. The concentrations of protein and precipitants increase as the two equilibrate, if appropriate solutions are used the crystal will grow.
Can be performed in either the hanging drop or sitting drop set up.

Question 18

Other than vapour diffusion, what other method can be used for protein crystallisation?

Answer 18

Microbatch, this uses a semi permeable membrane in which small molecules and ions can cross but large polymers cannot. By establishing a gradient the system slowly equilibrates and supersaturation occurs.

Question 19

What is the basic building block of a crystal?

Answer 19

The unit cell, contains proteins packed into a particular arrangement, which is repeated throughout the protein.

Question 20

What converts a diffraction pattern to a resolved crystal structure?
What converts a waveform or electron density to waves of different amplitudes and phases?

Answer 20

Fourier synthesis.

Fourier transformation.

Question 21

Resolution is quoted in terms of what and when is maximum resolution achieved?

Answer 21

It is quoted in terms of interplanar spacing. Maximum resolution is achieved when sinθ is largest. When this is largest, the crystal can be resolved to near atomic resolutions.

Question 22

What is triosephosphate Isomerase?

Answer 22

It is a glycolytic enzyme which catalyses the interconversion of DHAP and D-GAP. TIM has a high catalytic efficiency.

Question 23

How has the mechanism of TIM been studied?

Answer 23

Isotope exchange experiments,
pH activity dependence,
Affinity labelling,
X-ray crystallography.

Question 24

What is the main structure of TIM in solution and what is conserved between TIM?

Answer 24

The main structure of TIM is alpha/beta barrels in solution which act as a scaffold for active site residues. The active site residues are always on the C-terminal ends of beta strands. There is tremendous diversity in activity and little sequence homology but Glu165, His95 and Lys12 are absolutely conserved.

Question 25

How can the active site acids and bases be determined?

Answer 25

Residue (Gly165, His95 and Lys12) are conserved across species however this could suggest a structural role.
Crystal structure: look for residues in known active site,
Mutagenesis: mutate residues thought to be important and measure Km,
Conduct a pH profile of enzymatic activity.

Question 26

How was Glu165 identified as a catalytic base?

Answer 26

A pH profile found a residue with a Pka of 6.2 that was important for catalysis. E165D mutation was 1000 times less active, however it was a conservative mutation resulting in a 1Å change in the crystal structure. A transition state analog, phosphoglycohydroxyamate showed Glu165 was appropriately positioned to act as a base.

Question 27

How was His95 identified as a catalytic acid?

Answer 27

The transition state analog phosphoglycohydroxyamate was used and His95 was observed in a H-bond with it. The H95Q mutation results in a 400 fold drop in activity.

Question 28

Summarise the TIM mechanism.

Answer 28

Glu165 acts as a general base and helps transfer the C1 proton. A general acid (His95) helps transfer the hydroxyl proton by reducing the PKa of C1. Phosphate elimination is disfavoured by orbital elimination and by providing an apolar environment for phosphate.
Lys12 and the macrodipole of the alpha helix polarise the substrate carbonyl bond.

Question 29

How do enzymes function?

What is the Gibbs free energy effected by?

Answer 29

They function by lowering the Gibbs Free Energy. A large number of weak interactions between E and S provide the driving force for catalysis. Preferential stabilisation of the transition state reduces the Gibbs free energy and thus increases rate.
It is effected by changes in entropy and enthalpy.