Biocatalysis 2

Question 1

What symbol represents the turnover number?

Answer 1

K2 (Kcat) - rate of catalytic conversion

Question 2

What is kcat X Eo equivalent to?

Answer 2

Vmax

Question 3

What is Km a measure of?

Answer 3

The binding affinity

Question 4

What is the unit of activity?

Answer 4

1U = mmol product/min/mg E (per active site)

Question 5

What is the calculation for proficiency?

Answer 5

Kcat/Kuncat (rate of uncatalysed reaction)

Question 6

What is specificity a measure of?

Answer 6

Compares activity on two substrates

(kcat/Km)1 / (kcat/Km)2

Question 7

What is the benchmark of the enantiomeric ratio?

Answer 7

~100

Question 8

What is the Y intercept on a line-weaver burk plot?

Answer 8

The y intercept is 1/Vmax

Question 9

What is the X intercept on a line-weaver burk plot?

Answer 9

The X intercept is -1/Km

Question 10

What is the gradient of the line-weaver burk plot?

Answer 10

The gradient = Km/Vmax

Question 11

Why is there a large error rate in line-weaver burk plot?

Answer 11

There is a large error rate because of the reciprocal values – small changes in substrate concentrations, produced large error.

Question 12

What is the benefit of the Eadie-hofstee plot over the line-weaver burk plot?

Answer 12

The Y axis is not a reciprocal

Question 13

What is the benefit of the Hanes woolf plot over the Eadie-hofstee plot?

Answer 13

No longer has the substrate as the denominator hence the substrate error dependence is almost completely removed

Question 14

What is competitive inhibition?

Answer 14

Inhibitor competes for the active site.

Reaction rate is the same but interfers with binding kinetics hence Km changes and Vmax stays the same

Question 15

What is Non-competitive inhibition?

Answer 15

The substrate binds distal from the active site – allosteric –binds E or ES
Constant Km, but the Vmax changes

Question 16

What uncompetitive inhibition?

Answer 16

Binds the ES complex and prevents productive reaction

Vmax gets smaller and Km gets bigger

Question 17

What is the mathematical link between the temperature and the rate of the reaction?

Answer 17

Rate k = Ae-^(Ea/RT)

Question 18

What are the two predicted states in the two state model?

Answer 18

Folded or unfolded

Question 19

What is the equilibrium constant that connects these states?

Answer 19

K

K is a thermodynamic parameter, that relates to the free energy of unfolding.

Question 20

What is the melting point?

Answer 20

The temperature at which half the enzyme is unfolded

Question 21

What is the temperature when DeltaG is at maximum?

Answer 21

Ts

Question 22

In the 3 state model what are the 3 states?

Answer 22

folded, unfolded, denatured

E U D

Question 23

Which is measured experiemtnally?
a) Kdobs
b) K
C) k

Answer 23

Kdobs

K

Question 24

What is the equation for the half life of an enzyme?

Answer 24

Thalf = ln 2/ kdobs

Question 25

How is Kdobs determined?

Answer 25

Plot the remaining enzyme activity vs. time to determine the half-life Gradient is -kd

Question 26

What is the equation for remaining enzyme activity?

Answer 26

lnE/E0

Question 27

What is Enzyme Immobilisation?

Answer 27

Immobilising enzymes on a support to reduce unfolding by restricting movement

Question 28

What is coupling?

Answer 28

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

Question 29

What is entrapment?

Answer 29

Hiding Enzyme behind something/ in a pore

Question 30

What is volumetric productivity?

Answer 30

In the whole volume of the reaction (not specific). | Weight of Product Formed/Volume/ Time

Question 31

What is a monophasic reaction?

Answer 31

Substrate ad enzyme are in a single phase | solvent is miscible

Question 32

What is a biphasic reaction?

Answer 32

Enzyme is in an aqueous reaction and substrate in organic phase This involves a mass transfer hence stir to help diffusion

Question 33

What is a dispersed enzyme?

Answer 33

Freeze dried enzyme (in solid phase)

Question 34

Why do enzyme deactivate in solvents?

Answer 34

No hydrophobic driving force to remain folded | Also less active in solvents

Question 35

What are the two theories to explain why the original solution the lyophilised enzymes are from affects the function?

Answer 35

Some salts retain more water Fume silicia causes formation of smaller crystals - larger SA

Question 36

What is the B-factor?

Answer 36

A measure of ability to move from mean position

Question 37

What is the aim of medium engineering?

Answer 37

To produce a medium that improves enantiomeric ratio

Question 38

What laboratory tool might be used to regenerate cofactors?

Answer 38

electrodes | - REDOX reactions

Question 39

How might 1 enzyme be used to regenerate an co factor and still discourage the reverse reaction?

Answer 39

2nd substrate is introduced to regenerate the cofactor without having to perform the reverse reaction

Question 40

How might 2 enzymes be used to regenerate an co-factor?

Answer 40

Second enzyme converts a second substrate to a product to generate the co factor

Question 41

What is an issue with using 1 enzyme to regenerate the cofactor?

Answer 41

The enzyme is rate limited as it has to perform both reactions

Question 42

What is biotransformation?

Answer 42

Using cells to regenerate co-factors when multiple reaction steps and enzymes are required to do so.