Lecture 9 - Enzymes are essential for life Flashcards
Enzymes
Enzymes are biological catalysts – i.e. they increase the rate of a chemical reaction. (take reactants to products faster than they would on their own)
Most enzymes are proteins. (Catalytic RNAs, ‘ribozymes’ including ribosomes, are important exceptions.)
Enzymes, like all catalysts, do not change the free energy level of products and reactants.
A living cell enhances specific chemical reactions to create organisation until it is overtaken by death, chemical equilibrium and entropy.
Gibbs free energy
Life is not at equilibrium. For any chemical or biological process, the relative abundance of substrates and products is predicted by the Gibbs free energy…
ΔG < 0 Energy released; products dominate. Spontaneous reaction
ΔG > 0 Energy required; substrates dominate. Tends to be spontaneous in the opposite direction
ΔG = 0 At equilibrium; substrates and products at equal concentration.
Remember that thermodynamics and kinetics are independent I.e. energy is independent of rate
Synthesis pathways have a series of enzyme catalysed steps, keeps individual steps from equilibrium, can couple reactions (join a spontaneous and non-spontaneous reaction to give an overall delta G < 0
Activation energy
Energy required to each a transition state
Enzymes and catalysis explained
Enzymes catalyse reactions by lowering the activation energy
Cannot make a non-spontaneous reaction spontaneous
Does not shift equilibrium position i.e. same amount made but occurs faster
Enzymes do not change delta G they instead change the rate.
Enzymes speeds up reaction, doesn’t alter thermodynamics - changes size of the hump, not the relative start and end points
Energy needed to maintain cellular integrity
The overall Gibbs free energy (ΔG) has components of enthalpy (ΔH) and entropy (ΔS):
ΔG = ΔH - TΔS (T = absolute temperature) To favour the forward reaction (ΔG < 0) either enthalpy must
decrease (ΔH < 0) or entropy must increase (ΔS > 0).
Cellular integrity means a decrease in entropy in the cell, so energy from elsewhere is required. Enzymes control where and when energy is released to maintain the cell.
When temperature increases disorder also tends to increase
Why are some reactions slower?
Reactions pass through high- energy transition states. Activation energy (ΔG °‡) to reach the transition state determines rate. - height of the activation energy (height from reactants to transition state) dictates the kinetics from which the reactants will move to products. Higher the activation energy, less frequently they get over the bump and the slower the reaction is) Activation energy of back reaction = ΔG ° + ΔG °‡ At equilibrium, free energy change (ΔG °) sets ratio [Products]/[Reactants].
Transition state
The highest energy level required for a reaction to take place, it is the least stable state so if it is reached, the reaction will go to completion
High energy state containing partially formed and partially broken bonds
Cycle of enzyme catalysed reactions
1- Substrates enter active site; enzyme changes shape so its active site embraces the substrate (induced fit)
2- Substrates held in active site by weak interactions such as hydrogen bonds and ionic bonds
3- Active site (and R groups of its amino acids) can lower activation energy and speed up a reaction by acting as a template for substrate orientation, stressing the substrates and stabilising the transition state, providing a favourable microenvironment, participating directly in the catalytic reaction by providing ionisable amino acid side chains that facilitate the chemical reaction
4- Substrates are converted into products
5- Products are released
6- Active site is available for new substrate
Enzymes and catalysis
Enzymes catalyse thermodynamically favourable reactions by lowering the activation energy.
Rate enhancement differs from ΔG
Aldolase – very positive ΔG°, but big rate enhancement
Adenylate kinase – ΔG° near zero, big rate enhancement
Cleavage of DNA phosphodiester backbone: negative ΔG° – nonetheless stable for thousands of years uncatalyzed – catalyzed by ribonuclease A in less than a millisecond
Classes of enzymes
Oxidoreductases - redox I.e. transfer of electrons
Transferases - Transfer of a functional group
Hydrolases - Hydrolysis reactions (using H2O)
Lyases - Non-hydrolytic breaking or making of bonds that doesn’t use 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)
ATP hydrolyses examples that are very different
Muscle myosin uses energy from hydrolysis of ATP (general energy store) to drive muscle contraction
ATP synthase couples electrochemical gradient across the membrane to synthesise ATP.
Enzyme-substrate binding
Occurs at a specific site on the enzyme: the active site.
The active site:
o has amino acid side chains projecting into it.
o binds the substrate via several weak interactions. o determines the specificity of the reaction.
Substrate binds via
Weak interactions, types of enzyme-substrate bonds include : Ionic bonds Hydrogen bonds Van der Waals interactions Covalent bonds (rare, much stronger)
Weak interactions allow for specificity and reversibility
Active site
The active site: o has amino acid side chains projecting into it. o binds the substrate via several weak interactions. o determines the specificity of the reaction.
Highly specific for one reaction, particularly to the shape of transition state
Are specific i.e. there is molecular complementarity between enzyme and substrate
Types of enzyme-substrate bonds
Ionic bonds (a.k.a. salt bridges): o Make use of charged side chains (Asp, Glu, Arg, Lys).
Hydrogen bonds:
o Side chain or backbone O and N atoms can often act as hydrogen bond donors and acceptors.
Van der Waals interactions:
o Between any protein and substrate atoms in close proximity; weakest of the interactions, but abundant.
Covalent bonds:
o Relatively rare; much stronger than the other bonds.
Specificity
Geometric - the active site and the substrate must have a similar shape
Stero - if the active site is asymmetric, the enzyme can distinguish between identical functional groups on the substrate
Two models for enzyme-substrate binding
Lock and key model
Induced fit model
Lock and key model
The shape of the substrate and the conformation of the active site are complementary to one another
Induced-fit model
The enzyme undergoes a conformational change upon binding to substrate. The shape of the active site becomes complementary to the shape of the substrate only after the substrate binds to the enzyme.
At the moment, induced fit model is supported by a lot of evidence. For example, some enzymes can catalyse reactions with more than one substrate, but these different substrates are still similar in shape.
Enzyme is complementary to substrate
Many, weak interactions ensure specificity and reversibility:
o Several bonds are required for substrate binding - specificity.
o Weak bonds can only form if the relevant atoms are precisely positioned.
o Weak bonds allow reversible binding.
If you had multiple strong interactions then you would bind very strongly and never release it and if you want this enzyme to do its job over and over again then this won’t work as you have to not only bind the right thing as a reactant but you also have to release the right product.
Molecular complementarity between enzyme and substrate is critical.
Optimal binding is not too tight
Hill up to the transition state is higher if the substrate binds tightly to the enzyme
Tight binding= energetically favourable = deep energy well, deeper the enzyme-substrate complex well, the tougher it is to get over the transition state and to the products
How is activation energy lowered?
1- Ground state destabilisation.
2- Transition state stabilisation (bring down the hill).
3-Alternate reaction pathway with a different (lower-energy) transition state.
(1) and (2) can be achieved the same way: by having an active site that has shape/charge complementarity to the transition state, not the substrate.
Transition state analogues
Have a chemical structure that resembles the transition state
Strategies for catalysis
1- Acid-base catalysis
2- Covalent catalysis
3- Redox and radical catalysis (metal ions)
4- Geometric effects (proximity and orientation)
5- Stabilisation of the transition state
6- Cofactors with activated groups, e.g. electrons, hydride ion (H-), methyl groups (CH3), amino groups (NH2).
• This list is not exhaustive, but explains much enzyme activity.
• These are not exclusive: many enzymes use more than one from the list.
For two molecules to react …
They need to be close together AND in the right orientation
What drives covalent catalysis?
Nucleophilic attack on an electrophile dives covalent catalysis
Good electrophiles include bigger bonds such as I, Br and Cl
Good nucleophiles include OH-, negative, nitrogen, lone pairs (electrons to donate)
Nuclephilic attack requires correct orientation and ionisation (want more electrons)
Cofactors
Many enzymes require other non-protein factors to help them catalyse reactions.
Two classes of cofactor:
o Metal ions
o Coenzymes
Two classes of cofactors
Metal ions e.g. Zn2+
- Lewis acids (electron acceptors) so participate in acid base reactions
- Form coordination compounds, which can position the reactants
Coenzymes
- small organic molecules
- cosubstrates
- carrier (electrons, atoms or functional groups)
- derived from vitamins
Metal ion catalysis
More than a third of known enzymes require metal ions.
Specific coordination geometry orients substrates.
As Lewis acids, metals accept an electron pair to polarise H2O and functional groups.
Transfer electrons in oxidation-reduction reactions.
Mg2+
Zn2+
Fe2+ or Fe3+
Mn3+ or Mn4+
DNA polymerase; hexokinase; pyruvate kinase
Alcohol dehydrogenase; carbonic anhydrase
Cytochrome oxidase; peroxidase
Photosystem II
Hexokinase
Hexokinase uses Mg2+ as a cofactor - stabilising for a nucleophilic attack
Establishes orientation of phosphates of ATP by octahedral coordination of Mg2+ ion.
‘Electron withdrawing’ Lewis acid: stabilises electrons on oxygen, making phosphorous a better electrophile.
Nucleophile here is the hydroxide off of glucose, attacks the phosphate with its lone pair of electrons, to make it a better electrophile, magnesium sits with its 2+ charge and pulls electrons on to the oxygens so that they will be very well stabilised in the negative state. Makes oxygens better electrophiles and more susceptible to the attack by the hydroxide.
Coenzymes
Are small organic molecules. Are co-substrates. Are carriers (of electrons, atoms, or functional groups). Are often 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.
Will a biological process occur spontaneously if ΔG > 0? Why/Why not?
If gibbs energy is positive the process will not occur spontaneously but may occur in the reverse direction spontaneously. As energy is required for the process to occur
Do enzymes alter the equilibrium of a reaction?
Do enzymes alter the equilibrium of a reaction?
In short, enzymes do not change the equilibrium state of a biochemical reaction. ΔG0 and Keq remain the same. Instead, the enzyme reduces the activation energy needed for the reaction to proceed, and thus increase the rate of reaction. … And as such, they catalyze reactions in either direction!
Does an enzyme bind the substrate or the transition state more tightly
Substrate
How is the progression of a reaction through the transition state affected by the presence of an enzyme?
Activation energy is lowered, increasing the rate of reaction
Why do we need a formal classification system for enzymes?
enzymes are principally classified and named according to the reaction they catalyse. The chemical reaction catalysed is the specific property that distinguishes one enzyme from another, and it is logical to use it as the basis for the classification and naming of enzymes.
How do metal ions aid enzymes in metal ion catalytic mechanisms (illustrate with an example)?
More than a third of known enzymes require metal ions.
Specific coordination geometry orients substrates.
As Lewis acids, metals accept an electron pair to polarise H2O and functional groups. Transfer electrons in oxidation-reduction reactions.
How do coenzymes aid in enzymatic reactions?
Enzymes are proteins that speed up chemical reactions and often require cofactors to function. Non-protein organic cofactors are called coenzymes. Coenzymes assist enzymes in turning substrates into products. They can be used by multiple types of enzymes and change forms.
