Lecture 11 - Enzyme Rate (Michaelis-Menten vs Allosteric Enzymes) Flashcards
Enzymes catalyse thermodynamically favourable
reactions by
lowering the activation energy.
To model of enzyme catalysis, we use a very simple system in which an
enzyme, E, converts a single substrate, S, to a single product, P, that is instantly released.
is the conversion of enzyme, E, converts a single substrate, S, to a single product, P, that is instantly released reversible or irreversible?
irreversible
Relative speeds of k1 and k-1 define
how tightly
substrate binds.
The rate of catalysis, k2, relates to
energy of activation for the transition state.
‘Steady state’ refers to
time during which [ES] does not change.
why is ES complex necessary for reaction?
so [ES] at any time will govern the rate.
‘progress curve’ measures
appearance of product (or disappearance of substrate) with time at steady state.
Following the progress of an enzyme catalysed reaction
we measure…
initial reaction velocity (rate) i.e.
near time zero – symbol is V0
(or Vi or Vinit).
What is The effect of enzyme concentration on reaction rate when there is sufficient excess of substrate?
amount of enzyme increased, the rate of reaction increases.
when substrate is in excess what is proportional to [E] enzyme concentration?
Vo, initial velocity,
As [S], concentration of substrate, is increased, the initial rate V0…
increases in a linear way at first.
what does the hyperbolic curve show on V0 vs [S] graph?
Enzyme properties.
initial rate (V0) increase linear
Enzyme actives sites are occupied. rate of reaction stops increasing.
what can be identified on a V vs [S] curve?
Two kinetic parameters
Vmax
maximum velocity possible,
when [S] = ∞.
Km
Michaelis constant
substrate concentration at which Vobs = Vmax /2.
The Vobs vs. [S] curve is described by
Michaelis-Menten equation:
Michaelis-Menten equation:
Vobs = Vmax [S] / Km + [S]
How to determine enzyme kinetic parameters?
Michaelis-Menten behaviour
Michaelis-Menten model and assumptions
- Product is not converted back to substrate.
- Haldane’s steady state assumption: the rate of ES
formation equals the rate of its breakdown; that is
d[ES] / dt = 0
- Measuring initial rate ensures [S] does not change
significantly (and [S] is much greater than [E]).
Michaelis-Menten model and assumptions
what is Haldane’s steady state assumption?
d[ES] / dt = 0
Haldane’s steady state assumption: the rate of ES
formation equals the rate of its breakdown;
ES complex converts to E + P with
first order kinetics
Single molecule events, like radioactive decay, occur with a set probability, giving
first order kinetics.
why will ES ® E + P step follow 1st order kinetics?
If each ES complex has the
same chance of going
through the transition state,
When the Michaelis-Menten model fits
Some assumptions:
- All ES complexes have same rate of reaction.
- [S] is in vast excess to [E].
- Haldane’s steady state assumption: the rate of ES
formation equals the rate of its breakdown. - Initial rate is measured. That is, early enough that [S] does not change significantly.
- The reverse reaction does not occur.
do Cooperative enzymes follow Michaelis-Menten Equation?
No
what curve does Vobs vs. [S] plot show?
Sigmoidal
allosteric enzymes examples?
Aspartate transcarbamylase (ATCase)
phosphofructokinase
Cooperative enzymes do NOT follow Michaelis-Menten Equation.
Vobs vs. [S] plot
• Respond more steeply to
intermediate changes in [S].
• Evolve at regulatory points
in metabolic pathways.
• Recall haemoglobin.
Allosteric enzymes respond to
effectors binding away from the active site.
Allosteric enzymes Binding accompanies
change of shape,
turn changes enzymatic activity.
Allosteric enzymes have
multiple subunits and display cooperative behaviour.
Both cooperativity and allostery depend on the
enzyme switching between active and inactive forms.
Allosteric ATCase controls entry
to pyrimidine biosynthesis.
what is the first ‘committed step’ in making CTP, UTP and TTP?
Aspartate transcarbamylase (ATCase)
CTP
inhibits ATCase
ATP (a purine nucleotide)
activates ATCase, helping
to balance production.
ATCase shows most cooperativity in
presence of inhibitors.
V0 vs Aspartate plot
what is the curve where ATCase in presence of CTP?
Sigmoidal
V0 vs Aspartate plot
what is the curve where ATCase in presence of ATP?
Hyperbolic
Almost fits Michaelis-Menten model
ATCase includes
dimer of trimers and trimer of dimers
what do Regulatory dimers bind and control orientation of catalytic timers?
CTP or ATP
Catalytic trimers
shift orientation and conformation.
Active sites sit at
interfaces within trimers.
ATCase activation
what state do Top and bottom trimers bind in?
T-state,
distorting active site.
ATCase activation
what happens when Disengage in R-state?
allows substrate binding sites to come closer.
what controls glycolysis
Phosphofructokinase
Phosphorylates fructose-6-phosphate (F6P) to
fructose bisphosphate.
Phosphofructokinase controls glycolysis.
Inhibited if cell has plenty of
ATP, i.e. when glycolysis is not needed for energy.
Phosphofructokinase controls glycolysis
Homotetramer is cooperative
when
inhibited by ATP or
phosphoenolpyruvate (PEP).
Phosphofructokinase conformations
T-state
more compact,
stabilised by PEP,
an abundant intermediate of glycolysis.
Phosphofructokinase conformations
R-state is stabilized by
substrate F6P and ADP.
Phosphofructokinase conformations
what swap positions in active site?
Arginine 162 and Glutamic acid 161
At any given time, some need
to turn on and others need to
drop out.
- Enzyme amount
- Allosteric control
- Cell location
- Proteolytic activation
- Post-translational modification (e.g. phosphorylation of serine).
pancreatic digestive enzymes
- Zymogens are secreted from the pancreas in inactive form.
- Cleavage by proteases in the gut produces active enzymes.
- Temporal and spatial control.
Zymogens are
secreted from the pancreas in inactive form.
Michaelis-Menten equation is based on
binding theory and simple chemical reaction rates.
Derivation depends on
assumptions which limit how the equation can be used.
Allosteric enzymes change
shape and activity.
If multimeric,
allosteric enzymes often are
cooperative.
Allosteric enzymes control
metabolic pathways.
