enzymes Flashcards
exergonic vs endergonic reactions
exergonic = releases energy
energy required at start less than energy released by end = overall release
endergonic = require energy
energy required greater than released = overall need
transition state
point of maximum energy requirement
needs to be reached before a reaction can occur
(top of graph)
activation energy
energy required to make the reaction happen
molecules must collide before they can react
collisions don’t always provide enough energy for reaction to happen so sometimes an external source is needed eg heat
lower activation energy = reaction happens quicker
enzymes lower activation energy
what is an enzyme?
globular proteins with a 3D tertiary structure
act as catalysts by lowering activation energy
provide an alternate transition state
reduces amount of energy needed to transform substrates into products
amount of energy released is the same just the reaction occurs quicker
enzyme specificity
substrate and enzyme must collide and bind
only bind to specific substrates due to specific active site shape
active site composed of 2 parts:
substrate binding site
- complementary to substrate
- lock and key and induced fit
catalytic site
types of specificity
absolute = only do one reaction on one substrate
bond = breaks specific bonds
group = enzymes that add specific groups to molecules
stereo - binding site binds specific parts of substrate in specific order
catalytic mechanisms (5)
proximity - enzyme brings substate molecules close together so they can react
orientation - brings substrates to correct relative orientation
strain/distortion - enzyme binds to molecule putting strain on bond making it easier for reaction to occur
acid base catalysis - charges on enzymes means protons donated or accepted
covalent catalysis - temporary covalent bond formed between enzyme and substrate
more than one mechanism can be used to catalyse a reaction
what is the turnover number?
number of substrate molecules that can be converted to products by 1 enzyme molecule in 1 second
effect of time on enzyme action
initial reaction velocity (initial rate)
start occurs quickly as more substrate so a greater chance of collision
high substrate to enzyme ratio
rate slow as substrate decreases until plateau
reaches equilibrium
no net change in amount of substrate or product
effect of pH on enzyme activity
change in pH effects electrostatic bonds in protein
changes 3D structure
cant bind and function properly
decreases enzyme activity
effects bonds in substrate so can’t bind = decreased activity
all have optimum pH where the work best
denatured in too high or low
how does pH effect enzyme structure
decreasing pH
carboxylic acid soup gains proton, loses negative charge and electrostatic bond broken
increasing pH
NH3+ loses proton, positive charge lost, bond broken
also effects charged groups and therefore bonds in substrate
alters shape and therefore decreased activity
effect of temperature on enzyme activity
increasing temperature = more energy
kinetic effect
molecules move and vibrate more
number of collisions increases
= increased rate of reaction
until certain temperature
= denature
vibrate too much and breaks bonds between molecules
may reform elsewhere
= shape changed
effect of substrate concentration on enzyme activity
if number of enzyme molecules remain constant,
low concentrations
substrate concentration determine rate of reaction
- not enough collisions, active sites not full so rate slowed
high concentrations
number of enzyme molecules determine rate
- not enough enzymes for substrate to bind to, active sites full so rate limited
first order kinetics
velocity directly proportional to substrate concentration
velocity dependant on substrate
zero order kinetics
velocity independent of substrate
number of enzyme molecules determine velocity instead
hyperbolic curve
typical curve on graph of typical enzymes
Michaelis-Menten kintetics
Michaelis-Menten kinetics
Km = indicator of affinity of an enzyme for its substrate
high Km = low affinity
- ES complex less stable
- enzyme doesn’t bind to substrate well
- more substrate needed to stabilise therefore high Km
low Km = high affinity
- ES complex stable at lower concentrations
- enzyme binds to substrate well
plateau on graph only reached at very high concentrations
hard to reach in lab conditions
so Lineweaver-Burke used
what is Vmax?
maximum rate of reaction
where curve plateaus
determined from mm of curve
what is the Michaelis constant (Km)
substrate concentration at half Vmax (maximum rate of reaction)
Lineweaver Burke
plot the reciprocals of Vmax and Km
produces a straight line that can be extrapolated
however prone to high error
hard to accurately determine concentration when working at low concentrations
Lineweaver-Burke equations
gradient = Km over Vmax
1/Vmax = line intercepts Y axis
-1/Km = extrapolated back to x axis
what is an inhibitor?
molecules that reduce the rate of an enzyme catalysed reaction
can be irreversible or reversible
reversible = competitive and non-competitive
irreversible inhibitors
binds irreversibly to enzyme
form strong covalent bond
bind to amino acid side chain near active site
- commonly serine or cystine
inactivates enzyme by preventing substrate binding
reversible inhibitors
can be removed
includes
competitive
- bind to active site and compete with substrate
non-competitive
- bind to another site
what is a competitive inhibitor? (r)
compete for access to active site
similar structure to substrate
prevents binding of substrate
- doesn’t prevent catalysis
can be overcome by increasing substrate concentration = outcompete inhibitor (more likely to collide)
effects of competitive inhibitors
Km increases
forms enzyme inhibitor complex
therefore less ES complexes
amount of substrate required to reach half Vmax increases
more substrate needed to outcompete and form ES not EI complexes
Vmax remains the same
maximum rate unchanged
increase substrate concentration to outcompete inhibitor
always reach same Vmax despite inhibitor
what is a non-competitive inhibitor? (r)
bind at site other than active site
can occur before or after substrate binds
cont prevent substrate binding
- but prevent catalysis
cant be overcome by increasing substrate concentration
instead repeat dialysis to remove
enzyme inhibitor substrate complex made and changes shape of active site
binding of substrate not affect but cant catalyse reaction
effects of non-competitive inhibitors
Km remains the same
doesn’t effect binding of substrate
amount required to reach half Vmax remains the same
Vmax decreases
prevent catalytic act
maximum rate of enzyme decreased
enzyme activators
increase rate of reaction
or needed for activity of enzyme
also called cofactors
eg coenzymes
coenzyme A or acetyl CoA
allosteric enzymes
composed of 2 or more subunits
subunit can be in active or inactive form
active = low affinity for substrate
inactive = high affinity for substrate
one subunit binds to substrate
initiates change of structure, stabilises active form (high affinity)
substrate then more easily binds to second subunit as stabilised in active not inactive
- co-operativity
create sigmoidal kinetics (not hyperbolic)
control of allosteric enzymes
each substrate has one or more effector binding sites
positive effector = allosteric activators - increases overall enzyme activity
bind to effector binding sites
stabilise active form, substrate binds more readily
increased activity
negative effector = allosteric inhibitor - decrease overall enzyme activity
bind on effector sites, stabilise inactive form
substrate binds less readily
decreased activity
sigmoidal curve
s shaped curve
lag phase
move from inactive (low affinity) to active (high) form
substrate binds to subunits and changes shape
allows substrate to bind more easily
effect of allosteric enzymes on sigmoidal curve
positive effector
curve moves left
Vmax reached quicker
increase in activity over
substrate concentration quicker
negative effector
curve more right
vmaz reached slower
increase in activity over
substrate concentration slower
