Lecture 9 - Enzymes Flashcards
Enzymes are central to life
Describe what enzymes are and do
catalysts - Increase rate of reaction
Proteins - Catalytic RNA, Ribozyme, Ribosome
Enzymes do not
change free energy level of products and reactants.
Enzymes Fight against entropy by…
Keeping cells organised,
control gradients,
control pH
Liver cells
Responds to insulin
Turn sugar into glycogen or
mobilise glycogen into sugar
ΔG = 0
At equilibrium
Substrates & Products equal concentration
is life at equilibrium?
no
ΔG < 0
Products dominate
Energy released spontaneous
- ΔG
Want to drive
ΔG > 0
Energy required
substrates dominate.
+ ΔG
what is needed to maintain cellular integrity?
energy
ΔG = ΔH - TΔS
Gibbs free energy
Enthalpy
Entropy
To favour forward reaction (ΔG < 0)
Enthalpy decrease (ΔH < 0)
Entropy increase (ΔS > 0)
Cellular integrity means
decrease in entropy (ΔS) in cell.
Energy required somewhere else.
Enzymes control
where and when energy is released to maintain the cell.
To keep reactions going
ΔG < 0
Negative
Kinetics
How quickly is it going to reach the equilibrium
timescale for life
- Reactions pass through high- energy transition states.
- Activation energy (ΔGo‡) required to reach transition state. determines rate.
• Free energy change (ΔGo)
sets ratio [P]/[S] at equilibrium.
• Activation energy of back
reaction = ΔGo + ΔGo‡
What determines rate?
Activation energy (ΔGo‡) required to reach transition state.
Reaction that favours products
Moves forward
Negative ΔG
How fast its going to get there is governed by the transition state
Higher the barrier / hill on graph Free energy vs Progress of reaction
Slower reaction
higher activation energy
Back reaction slower
Products favoured over reactants
enzymes lower
activation energy
How do Enzymes catalyse thermodynamically favourable reactions?
lowering the activation energy.
Catalyzed version by enzyme
Has lower activation energy
Faster reaction
Rate enhancement
differs from ΔG
Aldolase
+ΔG
big rate enhancement
Adenylate kinase
ΔG near 0
big rate enhancement
Cleavage of DNA phosphodiester backbone
-ΔG
Stable for 1000 years uncatalyze
Catalyze by ribonuclease A in less than a millisecond
‘Isozymes’ differ in
sequence but catalyse the same reaction.
Classes of enzymes
- Oxidoreductases (Redox)
- Transferases
- Hydrolases
- Lyases
- Isomerases
- Ligases
Transferases
Transfer of a functional group.
Hydrolases
Hydrolysis reactions (using H2O).
Breaks down peptide bonds
Eg protease
Burn ATP
Lyases
Non-hydrolytic breaking or making of bonds (not using H2O).
Isomerases
Transfer of atoms/groups within a molecule to yield an isomeric form.
Ligases
Join two molecules together (i.e. form a new bond; usually coupled to ATP cleavage.
Enzyme–substrate binding occurs at
Active site
Enzyme active site
has amino acid side chains projecting into it.
binds substrate via weak interactions.
determines specificity of reaction.
Enzyme–substrate binding example
Hexokinase binding glucose
Types of enzyme substrate bonds
- Ionic bonds
- Hydrogen bonds
- van der Waals interactions
- Covalent bonds
Ionic bonds
Interactions of + and - charges
salt bridges
Make use of charged side chains (Asp, Glu, Arg, Lys).
H bonds
Side chain or backbone O and N atoms act as h bond donors and acceptors.
Stabilise α helix, b strand, protein structures, protein substrate interactions
van der Waals interactions
Between any protein and substrate atoms in close
proximity
2 atoms close up to each other
weakest
Abundant
Covalent bonds
Strong bonds
Rare
2 models for enzyme substrate binding
Lock and key
Induced fit
Lock and key model
Complementary
No change in conformation required
Active site perfect shape for substrate to bind
Shape of substrate and conformation of active site are complementary
Induced fit
Enzyme undergoes conformational change upon binding to substrate.
Shape of active site becomes complementary to shape of substrate only after substrate binds to enzyme.
glucose and hexokinase binding model
Induced fit
Enzymes are
dynamic (not static).
Enzymes show
geometric and stereospecificity
If shape of active site
is asymmetric,
enzyme distinguish between identical groups on substrate.
two CH2COO- groups.
weak interactions ensure
specificity and reversibility:
specificity.
Several bonds are required for substrate binding
Weak bonds can only form
relevant atoms are precisely positioned.
Molecular complementarity between enzyme and substrate is
critical.
How is Activation energy (ΔGo‡) lowered?
- Ground state destabilisation.
- Transition state stabilisation.
- Alternate reaction pathway with a different (lower-energy) transition state.
Ground state destabilisation and Transition state stabilisation is achieved by?
having an active site that has
shape/charge complementarity to transition state, not substrate.
Strategies for Catalysis not exhaustive and exclusive
- Acid-base catalysis
- Covalent catalysis
- Redox and radical catalysis (metal ions)
- Geometric effects (proximity and orientation)
- Stabilisation of the transition state
- Cofactors with activated groups,
Cofactors with activated groups examples
electrons, hydride ion (H-), methyl groups (CH3), amino groups (NH2).
For two molecules to react they need to be:
close together
In right orientation
Proximity and orientation
what drives covalent catalysis?
Nucleophilic attack on an electrophile
Electrophiles
Protons (H+)
Metal ions
Carbonyl carbon atom
Cationic imine (Schiff base)
Example of nucleophilic attack; requires
correct orientation and ionisation.
Cofactors
Non protein factors
Help enzymes catalyze reactions
2 classes of cofactor
Metal ion
Coenzymes
Metal ion catalysis
Specific coordination geometry orients substrates.
As Lewis acids, metals accept an electron pair to polarise
H2O and functional groups.
Transfer electrons in redox reactions.
Enzymes that use Mg2+
Hexokinase
DNA polymerase
Pyruvate kinase
What does Hexokinase use as a cofactor?
Mg2+
Hexokinase uses Mg2+ as a cofactor and establishes
orientation of phosphates of ATP by octahedral coordination of Mg2+ ion.
Hexokinase
Electron Withdrawing Lewis acid
stabilises electrons on
oxygen, making phosphorous a better electrophile.
Hexokinase
Uses ATP
Has Mg2+ which binds to phosphate of ATP to help establish geometry and withdraw electrons as a lewis acid
Electron withdrawing lewis acid
Coenzymes
- small organic molecules.
- co-substrates.
- carriers (of electrons, atoms, or functional groups).
- derived from vitamins.
Pyruvate dehydrogenase
Provides acetyl-CoA in aerobic conditions
Multienzyme complex composed of 30 copies of enzyme E1, 60 copies of E2 and 12 copies of E3, each with cofactors.
Net reaction is an oxidative decarboxylation.
Many cofactors of pyruvate dehydrogenase
CoA (coenzyme A) FAD NAD+ TPP Lipoic acid
what dictates speed of reaction?
Activation energy of transition state
What lowers activation energy?
Enzymes
Enzyme Active sites are
highly specific for one reaction, particularly to shape of transition state.
Many enzymes require
cofactors which confer specific abilities, e.g. redox activity.
