Biocatalysis 2 Flashcards
What symbol represents the turnover number?
K2 (Kcat) - rate of catalytic conversion
What is kcat X Eo equivalent to?
Vmax
What is Km a measure of?
The binding affinity
What is the unit of activity?
1U = mmol product/min/mg E (per active site)
What is the calculation for proficiency?
Kcat/Kuncat (rate of uncatalysed reaction)
What is specificity a measure of?
Compares activity on two substrates
(kcat/Km)1 / (kcat/Km)2
What is the benchmark of the enantiomeric ratio?
~100
What is the Y intercept on a line-weaver burk plot?
The y intercept is 1/Vmax
What is the X intercept on a line-weaver burk plot?
The X intercept is -1/Km
What is the gradient of the line-weaver burk plot?
The gradient = Km/Vmax
Why is there a large error rate in line-weaver burk plot?
There is a large error rate because of the reciprocal values – small changes in substrate concentrations, produced large error.
What is the benefit of the Eadie-hofstee plot over the line-weaver burk plot?
The Y axis is not a reciprocal
What is the benefit of the Hanes woolf plot over the Eadie-hofstee plot?
No longer has the substrate as the denominator hence the substrate error dependence is almost completely removed
What is competitive inhibition?
Inhibitor competes for the active site.
Reaction rate is the same but interfers with binding kinetics hence Km changes and Vmax stays the same
What is Non-competitive inhibition?
The substrate binds distal from the active site – allosteric –binds E or ES
Constant Km, but the Vmax changes
What uncompetitive inhibition?
Binds the ES complex and prevents productive reaction
Vmax gets smaller and Km gets bigger
What is the mathematical link between the temperature and the rate of the reaction?
Rate k = Ae-^(Ea/RT)
What are the two predicted states in the two state model?
Folded or unfolded
What is the equilibrium constant that connects these states?
K
K is a thermodynamic parameter, that relates to the free energy of unfolding.
What is the melting point?
The temperature at which half the enzyme is unfolded
What is the temperature when DeltaG is at maximum?
Ts
In the 3 state model what are the 3 states?
folded, unfolded, denatured
E U D
Which is measured experiemtnally?
a) Kdobs
b) K
C) k
Kdobs
K
What is the equation for the half life of an enzyme?
Thalf = ln 2/ kdobs
How is Kdobs determined?
Plot the remaining enzyme activity vs. time to determine the half-life
Gradient is -kd
What is the equation for remaining enzyme activity?
lnE/E0
What is Enzyme Immobilisation?
Immobilising enzymes on a support to reduce unfolding by restricting movement
What is coupling?
Enzyme can be attached to a solid carrier or cross link the enzyme to itself/carrier
Enzyme is in solid state and substrate must move from aqueous state to solid
What is entrapment?
Hiding Enzyme behind something/ in a pore
What is volumetric productivity?
In the whole volume of the reaction (not specific).
Weight of Product Formed/Volume/ Time
What is a monophasic reaction?
Substrate ad enzyme are in a single phase
solvent is miscible
What is a biphasic reaction?
Enzyme is in an aqueous reaction and substrate in organic phase
This involves a mass transfer hence stir to help diffusion
What is a dispersed enzyme?
Freeze dried enzyme (in solid phase)
Why do enzyme deactivate in solvents?
No hydrophobic driving force to remain folded
Also less active in solvents
What are the two theories to explain why the original solution the lyophilised enzymes are from affects the function?
Some salts retain more water
Fume silicia causes formation of smaller crystals - larger SA
What is the B-factor?
A measure of ability to move from mean position
What is the aim of medium engineering?
To produce a medium that improves enantiomeric ratio
What laboratory tool might be used to regenerate cofactors?
electrodes
- REDOX reactions
How might 1 enzyme be used to regenerate an co factor and still discourage the reverse reaction?
2nd substrate is introduced to regenerate the cofactor without having to perform the reverse reaction
How might 2 enzymes be used to regenerate an co-factor?
Second enzyme converts a second substrate to a product to generate the co factor
What is an issue with using 1 enzyme to regenerate the cofactor?
The enzyme is rate limited as it has to perform both reactions
What is biotransformation?
Using cells to regenerate co-factors when multiple reaction steps and enzymes are required to do so.
