Enzymes (lectures 6,7&8) Flashcards
What are enzymes?
proteins
catalysts
show specificity
sensitive to changes in physical & chemical environments
Examples of protein function
structure contractile transport defence storage regulatory catalytic
What are tamiflu & relenza?
neuraminidase inhibitors
neuraminidase removes neurotic acid residues from the surface of host cells to ease the release stage of the virus lifecycle
a mutation in the active site leads to tamiflu resistance
What does penicillin do?
binds to the enzymes that make the bacterial cell wall
What is the equilibrium constant (Keq)
at equilibrium the ratio of the concentration of products over reactants
Keq = [C][D] / [A][B]
Keq is dependent on the energy values of reactants & products
Gibbs Free Energy
the equilibrium constant of a reaction is correlated with the energy released by the reaction
Keq is proportional to delta G
Free energy = - RT(lnKeq)
for a reaction to be feasible free energy < 0
the bigger the free energy change the further over the equilibrium constant is
transition states
is is thermodynamically feasible for a high energy molecule to convert to a low energy molecule so releasing energy
intermediate ‘transition’ states in chemical reactions have a higher energy than reactants
so energy is requires to generate this reaction
Activation energy
determines the reaction rate
all catalysts lower AE
Enzyme-Substrate complex
enzyme (E) forms a complex with the substrate (S) before catalysing the conversion to product (P)
E + S –> ES –> P + E
formation of the ES complex leads to the alternative transition state
catalysis mechanisms
Proximity - closeness of substrate molecules
Orientation - correct relative orientation of substrates
Strain/distortion - binding puts strain on bond making it easier for reaction to occur
Acid-base catalysis - H+ donated/accepted or OH- generated
Covalent catalysis - temporary covalent bond between E & S
Specificity
enzymes can only bind to certain substrates
due to interactions between specific structural regions on the E & S
can be explained in 2 ways:
1) Lock & key - binding site has complementary shape
2) Induced fit - contact between part of the binding site & the S induces a change in shape of the active site
Kinds of specificity
Absolute - only works on 1 molecule
Bond - works on a group of molecules but targets a specific bond
Group - targets a specific group
Stereo - very stereo specific
6 types of enzyme reactions
1) Oxidation/reduction - oxidise or reduce a molecule
2) Transferases - transfer a group
3) Hydrolases - add hydroxyl groups
4) Lyases - elimination reaction
5) Isomerases - change type of isomer
6) Ligases - create covalent bond
What is the turnover number (Kcat)?
The no. of substrate molecules that can be converted to product by 1 enzyme in 1 second
How can we measure enzyme activity?
Either measure:
1) Disappearance of substrate
2) Appearance of product
if either is coloured we can use a spectrophotometer
The effect of varying substrate concentrations
At low [S], the no. of substrate molecules present determines how fast the reaction takes place
As [S] increases there is more chance of it colliding with an enzyme & for catalysis to occur
As [S] increases no. of ES complexes increases
How is rate linked to [S] ?
Rate of reaction = activity = velocity = V
V is proportional to [S]
First order kinetics
At high [S] all the enzymes will have bound substrates so will be working at their maximum rate
What is the Michaelis-Menten equation?
V = Vmax[S] / (Km + [S])
Where:
V = rate at specified [S]
Vmax = max value of V attainable by the enzyme under given conditions
Km = Michaelis constant = [S] at half Vmax
What does the Michaelis-Menten equation show?
Km measures stability of the ES complex - indicates affinity of enzyme for its substrate
High Km = low affinity
Low Km = high affinity
Km is in units of concentration (M)
Vmax is the fastest rate at which the enzyme can work
Vmax only occurs at infinite [S]
What are enzyme inhibitors?
reduce the rate of reaction
can either be:
1) Irreversible
2) Reversible - competitive, non-competitive & uncompetitive
Irreversible inhibitors
binds irreversibly to the enzyme
usually bind via a covalent bond
bind to an amino acid side chain at or near the active site
commonly bind to either Ser or Cys
binding permanently inactivates the enzyme
usually prevents substrate binding
examples of irreversible inhibitors
DFP
Aspirin
Penicillin
Competitive inhibitors
compete with S for access to active site
often have similar structure to S
when bound to E stops S binding
can be overcome by increasing [S] until it outcomes inhibitor
Km increases but Vmax remains unchanged
Examples of competitive inhibitors
Tamiflu
Acarbose
Statins
Non-competitive inhibitors
bind away from the active site
modify the reaction rate
still binds substrate with some affinity
influences capacity to catalyse reaction
Km unaltered but Vmax decreases
Uncompetitive inhibitors
occurs with multi substrate reactions
not important for drug therapy
binds only to ES complex
Km decreases & Vmax decreases
When will pH decrease enzyme activity?
If overall structure of active site is altered
If group involved in binding to S changes charge
