Kinetics Flashcards
Give an example of an enzyme that catalyses a ping-pong reaction
flavoprotein enzymes - succinate dehydrogenase/complex II
- the FAD becomes FADH (modified enzyme)
What does the X-axis of a Lineweaver-Burk plot show?
-1/Km
What does the y-axis of a Lineweaver-Burk plot show?
1/Vmax
What is a non-competitive inhibitor and what does the LB plot look like for a non-competitive inhibitor?
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
What is a competitive inhibitor and what does the LB plot look like for a competitive inhibitor?
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
What is an uncompetitive inhibitor and what does the LB plot look like for an uncompetitive inhibitor?
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
What does the LB plot look like for a sequential bi-bi reaction mechanism?
lines converge at x-intercept
What does the LB plot look like for a ping-pong bi-bi reaction mechanism?
parallel lines
Why is V0 measured?
product accumulation can cause feedback inhibition
What is the steady state?
The rate of substrate consumption is equal to the rate of product production
the concentration of ES is constant
What two factors can influence Km?
Ph and temperature
What is the shape of the curve provided by M-M steady state kinetics?
rectangular hyperbola
What is the absorbance maxima of NADH/NADPH?
340nm
Give an example of a enzyme coupled assay?
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
Other than spectrophotometrically, what other method can be used to track product production or substrate utilisation?
Radioactively labelled substrates - produce radioactively labelled products
What is the pre-steady state?
The first few milliseconds of a reaction following mixing of substrate and enzyme
What can be determined by pre-steady state kinetics?
1) the slow step
2) the individual rate constants (association/dissociation constants) for the formation of intermediates
The rate of the steady state only shows the ______ step of the reaction?
slow
name three methods of determining the pre-steady state kinetics?
1) stop-flow
2) quench-flow
3) temperature jump
Describe a stop-flow apparatus
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)
Describe a quench-flow apparatus and why might it be used over a stop-flow?
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
What is a downside of quench-flow?
it needs to be repeated multiple times, quenching at different times during the reaction to build up a reaction profile.
what is dead time?
the time between the mixing of the reactants and the collection of data begins
What two conditions must be met in order for pre-steady state to be used to determine individual rate constants of intermediates?
1) the reaction must proceed slow enough (i.e. beyond the dead time)
2) the intermediates must have different diagnostic characteristics
What is the coloured product that can be used to tract chymotrypsin in the pre-steady state?
4-nitophenol
What is the reaction carried out by alcohol dehydrogenase?
ethanol + NAD+ –> acetaldehyde + NADH
What reaction product of alcohol dehydrogenase can be tracked in the pre-steady state?
NADH production at 340nm
What is the absorbance maxima of NADH still bound to the active site of alcohol dehydrogenase?
NADH bound to active site has absorbance maxima at 320nm
