enzymes Flashcards
(34 cards)
5 classes of enzymes
oxidoreductases -catalyse redox
transferases - transfer functional groups
hydrolases - catalyse hydrolysis
isomerases - inter-molecular rearrangement
ligases - ligate 2 molecules
reaction if delta G < 0
exergonic (spontaneous)
free energy of products is lower
release energy
reaction if delta G> 0
endergonic (not spontaneous)
only occurs if input significant energy
how do enzymes work?
going from S-> P requires going through a transition state with higher free energy than S or P
enzymes lower the free energy of transition state
(ΔG‡ = energy barrier associated with going from substrate to transition state)
do not change overall ΔG of reaction
2 energy sources that reduce activation energy
1) non-covalent interactions
2) rearrangement of covalent bonds, using catalytic triad
energy sources to reduce activation energy
- non-covalent interactions
weak interactions in ESC releases a small amount of free energy, called binding energy
- this stabilises ESC, reduces Ea and stabilises the transition state
energy sources to reduce activation energy
- rearrangement of covalent bonds
use of a catalytic triad (Asp, His, Ser) in enzyme active site
1 acidic, 1 basic and 1 nucleophillic
bond rearrangements to form a reactive intermediate
this stabilises transition state and lowers activation energy
4 ways binding energy causes catalysis?
- enzyme holds substrate in close proximity in right orientation (but does decrease entropy, which is less favourable)
- desolvation of substrate - displacement of water surrounding substrate via non cov interactions with enzyme (releasing water increases entropy)
- binding energy compensates for unfavourable free energy changes when substrate distorts
- change in conformation upon substrate binding to enable catalysis (induced fit)
what is Vo like at low substrate concentrations?
at low [S], linear increase in substrate concentration
there are many free active sites, so rate is proportional to [S]
(this can be explained by michealis-menten kinetics)
what is Vo like at high [S]
Vo plateaus
A.S. saturated, reach Vmax
Rate is now independent of [s]
(this can be explained by michealis-menten kinetics)
what are steady-state kinetics?
pre-steady state: enzyme is first mixed with an xs of substrate (so ESC builds up linearly)
steady state : ESC virtually constant as massive excess of substrate so all enzymes in ESC form [most of reaction]
Therefore, we analyse the reaction when its in steady state
what is relationship between enzyme, Km and substrate?
a single enzyme has a different Km for different substrates
diff enzymes have different Km for different substrates
is high or low Km most favourable?
what is Km dependent on?
lower Km means the better an enzyme can process a substrate
depends on: substrate, pH and temperature
how does the magnitude of Km affect the strength of binding?
high Km = requires a large [S] for A.S to be 1/2 filled, so enzyme has a low affinit for substrate = weak binding
low Km = high affinity for substrate = strong binding
what is Km
substrate concentration at which reaction rate is half Vmax (so essentially half the active sites are filled)
what is Kcat
number of substrate converted to product when the enzyme is fully saturated
methods of measuring Km and Vmax
older method = manipulate michaelis-Menten equation to form a straight line
recent method = measure Vo at many [S]
use curve fitting programs on a computer to plot Vo against [S]
mechanism of action of a competitive inhibitor
- compete with substrate for active site
- form enzyme-inhibitor complex (temporary)
- block active site -> no catalysis
- dissociate - enzyme can now catalyse the reaction
example of a competitive inhibitor
enzyme - alcohol dehydrogenase
alchohol dehydrogenase converts methanol to formaldehyde (very toxic)
ethanol is a competitive inhibitor of AD (ethanol is converted to acetylaldehyde)
this prevents formation of toxic formaldehyde
what is an inhibitor
molecule that interferes with enzyme catalysis
how does a non competitive inhibitor work?
- bind to free enzyme or ESC at the allosteric sitge
- changes 3D shape of the enzyme
- substrate can still bind but is not in optimal configuration or catalysis
- when inhibitor dissociates, catalysis occurs
example of non-competitive inhibitor
PEP -> pyruvate -> alanine
alanine is a non-competitive inhibitor of pyruvate kinase (PEP-> pyruvate)
PEP can still bind to EI complex, but pyruvate cannot form
mechanism of uncompetitive inhibitor?
- substrate binds to enzyme
- inhibitor binds to ESC (only binds ESC)
- forms enzyme-substrate-inhibitor complex
- can dissociate - enable enzyme to catalyse the reaction
