Enzyme Kinetics

Question 1

What is the pre-steady state?

Answer 1

Isolated enzyme mixed with substrate to build-up an enzyme-substrate complex

Question 2

What is steady state reached in?

Answer 2

micrometres per second

Question 3

Why do enzymes exist in the steady state most of the time?

Answer 3

Regulate metabolism due to a constant supply of substrate

Question 4

What happens when new material is produced?

Answer 4

Current material is destroyed

Question 5

concentration of enzyme (E) is ____ compared to substrate (S)

Answer 5

negligible (more substrate)

Question 6

Why do we measure the rate of reaction after 1 minute?

Answer 6

Steady state has been reached

Question 7

Vo follows saturation kinetics re ____

Answer 7

substrate

Question 8

At low s, the reaction rate is ______

Answer 8

linear

Question 9

At very high s, vo approaches _____

Answer 9

vmax

Question 10

What is this known as?

Answer 10

theoretical maximum rate at which enzyme can operate

Question 11

What is the michaelis constant and what is it a measure of?

Answer 11

Km

measure of the affinity of an enzyme for its substrate

Question 12

What does alpha’ measure?

Answer 12

change in vmax which affects km

Question 13

Formation of ES is _____ and _______

Answer 13

rapid and reversible

Question 14

ES is a ________ complex

Answer 14

non-covalent, all takes place in 2nd step

Question 15

What is kcat known as?

Answer 15

turnover number

Follows 1st order kinetics

Question 16

Km is close to Ks, therefore _____ kinetics are ________

Answer 16

M-M

operable

Question 17

When Km>Ks what happens?

Answer 17

dissociation of ES is significant in comparison to kcat (Briggs-Haldane kinetics)

Question 18

When Km

Answer 18

Long-lived intermediates exist after substrate binding

Question 19

Why do enzymes have different kcat and km values?

Answer 19

cellular enviroment

Question 20

By assuming substrate < Km we can derive a ____ _____ in the form of a ____ order reaction

Answer 20

new equation

2nd

Question 21

What are the disadvantages of the lineweaver-burk plot?

Answer 21

• Compression of data points with high (S) into small region
• Favours data with low (S)
• Eadie & Hofstee decides to multiply everything through by V0Vmax giving y=c-mx

Question 22

What are the advantages of the Eadie-Hofstee plot?

Answer 22

• All values of (S) are weighted equally

- Considered to be more accurate determination of Km

Question 23

What are the disadvantages of the Eadie-Hofstee plot?

Answer 23

• Time consuming

- Both axes depend on Vo introducing potential for experimental error

Question 24

What are the advantages of the Woolf-Hanes plot?

Answer 24

Quicker to obtain data than E-H

Question 25

What are the disadvantages of the Woolf-Hanes plot?

Answer 25

Km is intercept not slope, thus more prone to error than E-H

Question 26

What happens in a Lineweaver-Burk plot of a ternary complex?

Answer 26

- Several different LB plots - Each plot has held S2 constant - Lines intersect

Question 27

LB behaviour for a ping-pong mechanism?

Answer 27

- Same experimental set-up | - Each LB plot is parallel to one another

Question 28

Many drugs act via what inhibition?

Answer 28

Reversible inhibition

Question 29

What is the most common form of inhibition?

Answer 29

Competitive inhibition e.g. use of ethanol to treat methanol poisoning - Inhibits alcohol dehydrogenase catalysed formation of formaldehyde, preventing blindness

Question 30

What is uncompetitive inhibition?

Answer 30

- Inhibitor only binds to ES complex - Stabilises the complex making it harder for the substrate to react & leave - Never seen in isolation e.g. Lithium ion treatment of manic depression > uncompetitive inhibition of myo-inositol monophosphatase

Question 31

What does mixed inhibition involve?

Answer 31

Binds to E or ES

Question 32

What does alpha measure?

Answer 32

measure of the extent of change in Michaelis constant (a=1 for no inhibition)

Question 33

What do non-competitive inhibitors bind to?

Answer 33

Allosteric sites rather than the active site

Question 34

What do non-competitive inhibitors act as and give example

Answer 34

Enzyme switches e.g Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs) > HIV1

Question 35

``` . In which type of graph would you plot Vo on the x-axis and Vo/[S] on the y-axis? A Eadie-Hofstee B Hill C Lineweaver-Burk D Michaelis-Menten E Woolf-Hanes ```

Answer 35

A Eadie-Hofstee

Question 36

``` On a Lineweaver-Burk plot for uncompetitive inhibition the “drug” and “no drug” lines: A cross at the x-axis B cross at the y-axis C cross elsewhere D are parallel to one another E are perpendicular to one another ```

Answer 36

D are parallel to one another

Question 37

``` On a Eadie-Hofstee plot the point at the which the line of best fit crosses the y-axis indicates: A Km B -Km C Km/Vmax D Vmax/Km E Vmax ```

Answer 37

E Vmax