6 - Mechanism of enzyme catalysis Flashcards
What is a catalyst and what does it do?
Catalysts stabilise the transition state of a specific reaction.
A catalyst:
- Lowers activation energy increasing ROR.
Is not consumed during the reaction.
Does not affect the eq. position.
What is a protein?
A protein is a large organic compound made of amino acids arranged in a linear chain and folded into a 3D structure
How does an active site form on a protein?
Protein folding brings together side chains of various amino acids that may be far apart in the primary sequence into close juxtaposition forming an active site.
What are the 4 stages of an enzyme-catalysed reaction?
- Enzyme + substrate
- Enzyme-substrate (E-S complex)
- Enzyme-product (E-P complex)
- Enzyme + product.
What are the 5 properties of the active site?
- Positions the substrate molecules in the most favourable orientation for reaction
- Perfectly complementary to transition state.
- Amino acid side chains of the AS stabilise the electron distribution of the TS.
- The substrate is strained on binding with AS lowering Ea and increasing ROR.
- The products bind less tightly to the AS than the substrates so are released.
What non-covalent interactions occur between the substrate and the amino acid side chains of the enzyme?
ionic bonds:
- Acidic groups (Asp, Glu)
- Basic groups (Lys, His, Arg)
hydrophilic interactions:
- OH groups (Ser, Thr, Tyr) - SH groups (Cys) -
- Amide groups (Asn, Gln)
Hydrophobic interactions:
- Ala, Leu, Leu, Ile, Val, Met
Aromatic interactions:
- Aromatic groups (Phe, Try, Trp)
What are 3 ways in which reactive groups at the catalytic site surface catalyse a reaction?
- Donating or withdrawing electrons
- Stabilising or generating free radical intermediates
- Forming temporary covalent bonds
What are cofactors, 3 examples of what they can be and 3 specific examples?
- Metal group such as (Mg2+)
- Prosthetic group - covalently bound organic molecule (haem)
- Coenzyme - tightly but not covalently bound organic molecule (NAD)
What are enzymes called when they are catalytically active and when they aren’t?
Enzyme with prosthetic group/ coenzyme - holoenzyme (active)
Enzyme without prosthetic group / coenzyme - apoenzyme (inactive)
What reactions do oxidoreductases catalyse?
Oxidation and reduction
What reactions do transferases catalyse?
Transfer a chemical group from one substrate to another.
What reactions do hydrolases catalyse?
Hydrolysis reactions
What reactions do lysases catalyse?
Addition across a C=C bond
What reactions do isomerases catalyse?
Intramolecular rearrangements
What reactions do synthetases catalyse?
Formation of bonds between two substrates
What is enzyme activity and what is it measured in ?
Number of micromoles (µmol) of substrate converted per minute under standard optimised conditions at 30°C.
Enzyme unit (EU) = µmol min-1
What is specific activity and what is it measured in?
Measures enzyme purity - activity of enzyme per mg of total protein in the enzyme preparation.
measured in - µmol min-1 mg-1
What 3 factors must be fulfilled for a meaningful quantitative assay of enzyme activity?
- Measured at fixed [enzyme]
- Measured at defined temperature and pH
- initial velocity V0 is measured.
What 5 factors affect enzyme activity?
- pH
- Temperature
- [Enzyme]
- [Substrate]
- Covalent modification of enzyme
Why does pH affect enzyme-catalysed reactions?
pH effects the ionisation state of amino acid side chain affecting the binding of the substrate.
What is enzyme denaturation?
Denaturation can occur at high temperatures and at extreme pH levels.
The extreme conditions lead to a loss of hydrogen bonding causing the enzyme to unfold resulting in a loss of activity.
Why does temperature affect enzyme-catalysed reactions?
Higher temperatures increase ROR
- Molecules move faster, greater collision chances, electrons gain Ea easier.
Temperature optimum depends on the time of incubation.
What effect does varying enzyme concentration have on enzyme-catalysed reactions?
Typically increasing enzyme concentration causes a linear increase in product formation.
In some situations the enzyme can associate or dissociate into a different form (monomer or dimer) which may be more or less active at different enzyme concentrations.
This can cause the linear increase to become curved - either increasing faster or dropping off at higher concentrations.
What effect does varying substrate concentration have on enzyme-catalysed reactions?
An increase in substrate concentration increases the rate of reaction until the enzyme becomes saturated at which point the ROR begins to level.
