Enzyme Kinetics Flashcards
how do enzymes have implications in disease
small changes in their abundance, efficiency or distribution
why can enzymes be used in lab to diagnose and develop therapeutic drugs
enzymes control well defined chemical reactions
role of enzyme
catalysts
convert specific substrateto product
more substrate = more chance of enzyme working on it = more product
what is saturation
increasing substrate conc is no longer the limiting factor as all active sites
what is Vmax
max rate at which enzymes can convert substrate to product per sec
what is Km
50% of max rate enzymes convert substrates to product
what model is used to explain the relationship between km and Vmax
Michealis-Menten
[ES] = K1[E][S]/(K-1+K2) constants on one side of the reaction
combination of rate constants
what is the activation barrier
point in reaction where energy is at its highest, enzyme substrate complex is leas stable and if barrier isn’t overcome, enzyme-substrate complex can go backwards
What are the rate constants in and enzyme substrate reaction
K1, K-1, K2
how are Vmax and Km measured
measure initial velocity Vo at a known substrate conc
Repeat at increasing substrate conc
what is the Michaelis constant Km equivalent to
substrate concentration where initial reaction is half maximal
reaction velocity never quite reaches true Vmax
what does the michaelis-menten equation describe
rate of catalysis as a function of substrate concentration
Velocity = max velocity/50% velocity + substrate
what is used to determine Vmax and Km
Michaelis-equation
can be written as
1/v = Km/Vmax x 1/s + 1/vmax
what is the line weaver-burk plot
accurate determination of Vmax and Km
we plot 1/v against 1/s
what is Vmax on line weaver-burk plot
intersection of straight line with Y axis
what is Km on line weaver-burk plot
intersection with x axis
-1/Km
what is the conc of substrate at the x intercept
infinity
what is Vmax
maximum velocity of reaction
what is Km
conc in moles of substrate which gives 1/2 Vmax
Km = [S] at 0.5 Vmax
when substrate concentration becomes smaller what do enzymes with steeper graphs do
work better at lower substrate concentrations as they are good at finding substrate and converting it to product
if slope is gradual enzyme doesn’t work well with low substrate concentration as more sensitive to changes in substrate conc
role of hexokinase
catalyses first reaction in glycolysis
works fast even when substrate conc is low
what does it mean if an enzyme has a high Km
sensitive to changes in substrate concentration
does hexokinase in red blood cells have a high or low Km
low Km = 0.05mM
Low Km maintains energy production in rbcs by glycolysis even if glucose falls
what is normal fasting blood glucose
5mM
what 2 enzymes catalyse glucose + ATP -> glucose-6-phosphate + ADP
Glucokinase - High Kw
Hexokinase - Low Kw
why does glucoskinase have a high Kw
enables glucose sensing, homeostasis
abundant in liver is regulated by insulin
excess blood glucose is metabolised
loss of activity - type 1 diabetes
oxygen sensors and what they are regulated by
propel hydroxylases
regulated by transcription factor HIF (hypoxia inducible factor)
what happens when prolyl hydroxylases are activated
genes for surviving hypoxia stitched on
rbc synthesis
blood vessel growth
anaerobic survival pathway
substrate of proline hydroxylases
oxygen to regulated HIF transcription factor
proline hydroxylase have a high Km for oxygen what does this mean
they aren’t good at finding substrate
therefore work as good receptors
2 types of reversible inhibition
competitive - inhibitor binds to active (catalytic) site and blocks substrate access - orthosteric inhibition (at same time)
Non competitive - inhibitor binds to allosteric site (not the catalytic centre) and inhibits enzyme by changing its conformation
example of irreversible inhibition
non-competitive
involved formation or breakage of covalent bond in enzyme complex
does competitive inhibition change Vmax or Km
Vmax does not change (y intercept) Km varies (x intercept)
give example competitive inhibition in the clinic
methanol poisoning
methanol is substrate for alcohol dehydrogenase (ADH), causes tissue damage/blindness by conversion to formaldehyde and drives metabolic acidosis
ADK Km for ethanol 20x greater so patient treated with ethanol
does non-competitive inhibition affect Vmax or Km
Km is the same (x intercept, -1/Km) Vmax varies (y intercept, 1/Vmax) Vmax is lower enzyme finds it more difficult to find substrate
what is feedback inhibition
inhibition of rate limiting enzymes by end product - allosteric control
end product is an inhibitor to an enzyme earlier in the pathway
avoids build up of intermediates in pathway
what type of curve do allosteric enzymes have
Sigmoidal curve (not hyperbola) they do NOT follow Michaelis-Menten kinetics
what are allosteric enzymes controlled by
allosteric inhibitor (curve lower) allosteric activator (strap higher and steeper)
in allosteric enzymes are Km and Vmax affected
Yes
example of allosteric regulation
binding of oxygen to haemoglobin
positive cooperatively
controlled by - H+, CO2, 2,3 bisphosphoglycerate (glycolysis side product)
my does myoglobin not show co-operativity
haemoglobin more sensitive to changes in concentration than myoglobin
what is Vo
initial reaction velocity at known substrate conc
what is K1
reaction form enzyme to enzyme substrate complex
what is K2
reaction from enzyme-substrate complex to product
what is K-1
back reaction from enzyme substrate complex back to enzyme
why can Vmax and Km not be measured accurately from hyperbolic plot of V/S (why double reciprocal is used)
the kinetics are not linear so reaction velocity never quite reaches true Vmax
in line weaver-burk complex enzymes with high Km will tend towards zero intercept on X axis
True
what is the Michaelis-Menten Equation
Vo = Vmax [S]/Km [S]
line weaver-burk straight line equation
1/Vo = Km/Vmax x 1/[s] + 1/Vmax
how do competitive inhibitors work
Form EI (enzyme inhibitor complex) + substrate which cannot react K1 step
how doe uncompetitive inhibitors work
forms ESI (enzyme substrate inhibitor) w cannot react
what is a non-competitive inhibitor
mixed
can bind to E forming EI
or ES forming ESI
what happens to line weaver burke slope as you increase competitive inhibitor
slope gets steeper as inhibitor increases
so if no inhibitor present slope more gradual
y intercept doesn’t change
increase in Km
Vmax unchanged
what happens to line weaver burke slope as you increase uncompetitive inhibitor
all 3 lins have same slope but different yintercepts
increase in inhibitor = decrease in Vmax
enzyme works at low substrate concentrations
effect of increasing non-competitive inhibitor on line weave burke slope
increases in slope
increase in Km
decreased Vmax
a plot of V[s] will always produce a hyperbolic relationship
false not true for allosteric enzymes
