Kinetics

Question 1

Give an example of an enzyme that catalyses a ping-pong reaction

Answer 1

flavoprotein enzymes - succinate dehydrogenase/complex II

- the FAD becomes FADH (modified enzyme)

Question 2

What does the X-axis of a Lineweaver-Burk plot show?

Answer 2

-1/Km

Question 3

What does the y-axis of a Lineweaver-Burk plot show?

Answer 3

1/Vmax

Question 4

What is a non-competitive inhibitor and what does the LB plot look like for a non-competitive inhibitor?

Answer 4

non-competitive inhibitor bind to the enzyme at a site distinct form the active site

LB plot = convergence at x-axis as Km stays same but gradient steeper as Vmax decreases

Question 5

What is a competitive inhibitor and what does the LB plot look like for a competitive inhibitor?

Answer 5

competitive inhibitor binds to the enzyme at the active site

LB plot = cross at y-intercept because Vmax stays the same (can be reached with increased concentration) but Km increased as substrate less able to bind

Question 6

What is an uncompetitive inhibitor and what does the LB plot look like for an uncompetitive inhibitor?

Answer 6

Uncompetitive inhibitor binds to the enzyme-substrate complex and prevents catalysis

LB plot = parallel lines because Vmax decreased and Km decreased as high affinity as substrate bound

Question 7

What does the LB plot look like for a sequential bi-bi reaction mechanism?

Answer 7

lines converge at x-intercept

Question 8

What does the LB plot look like for a ping-pong bi-bi reaction mechanism?

Answer 8

parallel lines

Question 9

Why is V0 measured?

Answer 9

product accumulation can cause feedback inhibition

Question 10

What is the steady state?

Answer 10

The rate of substrate consumption is equal to the rate of product production
the concentration of ES is constant

Question 11

What two factors can influence Km?

Answer 11

Ph and temperature

Question 12

What is the shape of the curve provided by M-M steady state kinetics?

Answer 12

rectangular hyperbola

Question 13

What is the absorbance maxima of NADH/NADPH?

Answer 13

340nm

Question 14

Give an example of a enzyme coupled assay?

Answer 14

Hexokinase produces glucose-6-phosphate and ADP, neither of which are optically traceable so are coupled with the glucose-6-phosphate dehydrogenase enzyme that uses NADP+ to oxidise G6P and produce NADPH which can be tracked at 340nm using spectrophotometer

Question 15

Other than spectrophotometrically, what other method can be used to track product production or substrate utilisation?

Answer 15

Radioactively labelled substrates - produce radioactively labelled products

Question 16

What is the pre-steady state?

Answer 16

The first few milliseconds of a reaction following mixing of substrate and enzyme

Question 17

What can be determined by pre-steady state kinetics?

Answer 17

1) the slow step

2) the individual rate constants (association/dissociation constants) for the formation of intermediates

Question 18

The rate of the steady state only shows the ______ step of the reaction?

Answer 18

slow

Question 19

name three methods of determining the pre-steady state kinetics?

Answer 19

1) stop-flow
2) quench-flow
3) temperature jump

Question 20

Describe a stop-flow apparatus

Answer 20

enzyme and substrates are kept in two separate precision syringes and are mixed in the mixing chamber, which causes the stopping syringe to move towards and hit the trigger. This stops the further mixing of reactants and triggers the acquisition of data by the detector (a high precision spectrophotometer which measures the optical change)

Question 21

Describe a quench-flow apparatus and why might it be used over a stop-flow?

Answer 21

might be used over a stop-flow if there is no observable optical change.

the enzyme and substrate are kept in two separate precision syringes and are mixed in the mixing chamber. A quenching agent is then added to stop the reaction and allow the collection and separation of products and substrates by chromatography

Question 22

What is a downside of quench-flow?

Answer 22

it needs to be repeated multiple times, quenching at different times during the reaction to build up a reaction profile.

Question 23

what is dead time?

Answer 23

the time between the mixing of the reactants and the collection of data begins

Question 24

What two conditions must be met in order for pre-steady state to be used to determine individual rate constants of intermediates?

Answer 24

1) the reaction must proceed slow enough (i.e. beyond the dead time)
2) the intermediates must have different diagnostic characteristics

Question 25

What is the coloured product that can be used to tract chymotrypsin in the pre-steady state?

Answer 25

4-nitophenol

Question 26

What is the reaction carried out by alcohol dehydrogenase?

Answer 26

ethanol + NAD+ –> acetaldehyde + NADH

Question 27

What reaction product of alcohol dehydrogenase can be tracked in the pre-steady state?

Answer 27

NADH production at 340nm

Question 28

What is the absorbance maxima of NADH still bound to the active site of alcohol dehydrogenase?

Answer 28

NADH bound to active site has absorbance maxima at 320nm