4. Enzyme Kinetics Flashcards
how do enzymes affect equilibrium?
they dont!! just speeds up the reaction
what does a reaction coordinate diagram show?
shows the reaction progress
what is the transition state?
barrier of energy that must be overcome to react product
what would happen if there was no transition state?
substrate wouldn’t be stable
how do enzymes affect transition state?
enzymes allow for a new transition state by lowering the energy barrier
they do this setting up an optimal substate position, weakening the bonds with environment, etc to allow smooth transition from substrate to product
what does the speed of enzymatic reaction rely on?
rate of reaction, determined by transition state
what does equilibrium depend on?
equilibrium depends on the energy difference between the substrate and product –> not affected by enzyme
describe what happens at transition state and how
substrate undergoes structural changes –> bonds breaking and making, unstable intermediate
what is activation energy? how do enzymes affect it?
energy required to induce the structural change of the substrate at the transition state
enzymes reduce it –> faster reaction
is there a specific direction a molecule is more likely to go thru?
no direction –> equally likely a molecule decays in either direction, i.e. substrate or product
what is the active site?
specialized pocket where reaction occurs
what is the environment of the active site? why?
hydrophobic to isolate itself from water
how does the active site work?
allows reactants to be properly positioned to easily attain their transition state by weakening bonds
(may participate in reaction)
does the active site have stronger interaction with transition state, substrate, or product?
transition state
what would be the result if an enzyme’s active site was complementary to the substrate?
substrate is too stabilized –> cannot reach transition state and enzyme acts as competitive inhibitor of the transition state –> no drive to catalyze anything
what would be the result if an enzyme’s active site was complementary to the transition state?
substrate can fit into enzyme but not perfectly –> can reach transition state naturally but FASTER!
What is the induced fit model of binding?
active site approximates the substrate, then properly binds once substrate is there
what is the selected fit model of binding?
active site changes conformation constantly and ligand stabilizes 1 conformation
describe the equilibrium of enzyme activity
E+S <–binding–> ES <–catalysis–> EP <–release–> E+P
each step is in equilibrium and has diff transition state
what do serine proteases do?
cleave peptide bonds
describe the 3 components of the serine protease active site
- Ser –> OH group acts as nucleophile that attacks peptide bond of substrate
- His –> accepts H from Ser OH to make nucleophile
- Asp –> H bonds with His, making N more electronegative
describe the alkaline phosphatase experiment
to see how fast substrates are consumed by phosphatase
if phosphatase consumes NPP (colourless), will produce NP (yellow) and can measure amount of yellow
what happens if you have more substrate per enzyme?
will saturate the enzyme and reach Vmax
what are the units of velocity?
mM/min
in the curve showing [product] vs time, what happens when the curve flattens?
substrates have been consumed –> enzyme stops working –> plateaus at an amount of products
what does the michaelis menten curve show?
velocity vs [substrate]
indicates that reaction speed will increase proportionally to [substrate] then saturate at Vmax
describe Vmax
independent of [substrate] –> there is more substrate than active sites available so increasing [substrate] has little effect
what is Michaelis constant?
Km = [substrate] and 50% Vmax = [substrate] when 50% of active sites are occupied
what does a high Km mean?
more substrate is required for a given reaction rate –> enzyme is less efficient, weak binding
what does a low Km mean?
less substrate is required for a given reaction rate –> enzyme is more efficient, high affinity
what do michaelis-menten equations assume?
assume formation/release of product is irreversible
how do you calculate Km?
Km = (k2 + k3)/k1
k1: E+S –> ES
k2: ES –> E+S
k3: ES –> E+P
what is the michaelis menten equation for V0
V0 = (Vmax*[S])/(Km+[S])
what is the problem with michaelis-menten plots? what do we use instead?
michaelis-menten plots may not show enough data points to determine whether curve has reached a maximum or not –> Vmax and Km likely inaccurate
use Double Reciprocal / Lineweaver-Burk
what is the double reciprocal/lineweaver burk plot?
uses reciprocals of velocity and reciprocals of [substrate]
how do you find the Vmax in the double reciprocal/lineweaver burk plot?
y-intercept
how do you find the Km in the double reciprocal/lineweaver burk plot?
slope or x-intercept
do enzymes only have 1 Km value?
no! can diff Km values for diff substrates
why may some enzymes have high Km even though it’s not efficient?
may be tailored to be slow to regulate certain conditions
what is competitive inhibition?
multiple substrates binding same site
how does a reaction change with competitive inhibitor?
velocities decrease and Vmax stays the same always, Km increases
how does increasing [substrate] affect competitive inhibition?
makes the inhibitor less effective –> outcompetes the inhibitor
what is uncompetitive inhibition?
requires substrate for inhibitor to bind and inhibit –> i.e. inhibitor needs ES complex
what happens if you increase [substrate] in uncompetitive inhibition?
there is no competition bc they bind diff sites
how do Vmax and Km change in uncompetitive inhibition?
both Vmax and Km decrease
what is non-competitive inhibition?
inhibitor binds in presence OR absence of substrate bc binds diff sites –> allows control by cell itself
how does inhibition change if you increase [substrate] with non-competitive inhibition?
no effect!
what is irreversible inhibition?
covalent bonds –> nothing goes on or off
what is a suicide inhibitor?
covalently attaches to enzyme and inhibits it –> tricks enzyme into thinking the substrate has bound
what is a transition state inhibitor?
mimics catalytic transition state with HIGHER AFFINITY than substrate or product (non-covalent, but quasi irreversible binding)
what are the 3 values to compare inhibitor effectiveness?
- Ki
- IC50
- EC50
describe Ki
EI <=> E+I
Ki = ([E][I]/[EI])
lower Ki = more potent inhibitor
describe IC50 and its equation
[inhibitor] that inhibits 50% of enzyme
IC50 = Ki + [E]/2
describe EC50
[inhibitor] that reduces cellular effect by 50%
what is the difference in studying IC50 vs EC50?
IC50 –> can look at pure enzyme + inhibitor
EC50 –> must look at whole cell system to see downstream effect
