Metabolism notes
L1 – ATP & Gibbs Free Energy – Enzyme Kinetics
Learning objectives
● How is ATP made? How does the cell know how much to make?
● Proteins consist of amino acids, but how are they made from sugar and other
nutrients?
● How does the cell synthesize the building blocks of cells?
Metabolism
● Is relevant for several diseases eg cancer, diabetes, metabolic syndrome
● is key for bio-based economy
Basic principles
● Metabolism is constrained by the laws of chemistry & physics → energy: the laws
of thermodynamics
● In healthy metabolic network, supply and demand of nutrients are balanced to
support growth or other vital functions → kinetics & regulation
● We take a quantitative approach, but physical and chemical principles should
always serve to understand biological function
Glucose: favourite fuel
● Brain and tumor cells particularly love glucose as a carb source
● Holds for many microbes as well, not all
● Other fuels for energy are used eg fats (long term storage: fasting)
● Proteins (muscles) can also be degraded to amino acids → another source for fuel to get ATP (unhealthy)
● Glycolysis: conversion of glucose → pyruvate (3C atoms, not yet CO2, not fully oxidised to glucose)
● Glycolysis in movement science:
○ Short and intense exercise depends primarily on glycolysis
○ Glycolysis can be upregulated 400x during a 100m sprint
● Glycolysis in cancer research
○ Diagnosis: radioactive analogue of glucose → PET scan shows highly glycolytic tumor lesions
○ Treating tumors: giving inhibitors of glycolytic enzymes & pathways, however targeting is tricky
(normal cells can be affected)
Glycolysis overview – details in tomorrow’s lecture
● To know: substrates of each reaction and the enzyme catalysing it
● No need to know the structures of the chemical compounds, but helpful to look at
● We start with a sugar with 6 Carbon atoms
● Stage 2: aldolase splits the compound into 2 compounds with each 3 Carbon atoms
○ DHAP and GA3P = can be interconverted very easily, reversible process, each can
be converted into pyruvate
○ 1 glucose → 2 pyruvate
● Glycolysis is the first phase of glucose catabolism
● ATP is a carrier of Gibbs energy (needed to start glycolysis)
○ Investment: 2 ATP
○ Gross yield: 4 ATP
○ Net yield: 2 ATP
● In stage 1: 2 molecules of ATP invested
● In stage 3: is done twice, therefore 2 X 2ATP produced
● Microorganisms have many variants of the canonical Embden-Meyerhof-Parnas pathway
(=glycolysis)
,Electron carriers
Biochemical redox reactions involve a limited number of electron
carriers. Reactive sites: where electrons are taken up. Conjugated
systems of alternative double bond and single bond → easily
shuffle around electrons. A few examples:
1. NADH is an electron carrier
● Many redox reactions in cells (electrons transferred
from 1 molecule to another)
● Often these reactions don't directly transfer electrons
to final acceptor
● As a stepping stone, they use NAD to store the electrons for a while → it makes it a universal currency of
electrons in the cell, just like ATP is a universal currency of energy
● If one reaction has excess electrons, it can just drop electron to NAD to produce NADH → NADH can then give
electrons wherever needed in the cell
2. NADP+
3. FAD
2 electrons come with a proton
● Electrons don't come alone
● They come with a H+ that then form a bond
● Carbon has 4 electron pairs (8 electrons) around it
● In these aromatic rings, the double bonds can just shift around the electrons and the
double bond goes to the Nitrogen (previously positively charged) → neutralised
NADH
● Large molecules like NADH needed to facilitate these biological processes because it
can absorb electrons and give them away easily
Alcoholic fermentation
● The formation of ethanol re-oxidizes NADH without an external electron acceptor → Fermentation
● Examples: yeast, goldfish, rice root.
● Some yeasts also ferment in the presence of O2, when glucose is abundant
The fate of pyruvate
● Lactate and ethanol are fermentation products.
● Fermentation is the process in which NADH generated in glycolysis donates its electrons to an internal, organic
electron acceptor (pyruvate).
● Under anaerobic conditions, fermentation is necessary to generate ATP.
● So pyruvate has 3 fates
a. Acetyl coa → Further oxidation
b. Fermentation → Lactate: in muscles or lactic acid bacteria
c. Fermentation → Ethanol
i. Fermentation: No o2, you need internal acceptor of electron = balance
of synthesis
Enzymes of glycolysis
1. Hexokinase
● Making a phosphate bond is energetically costly
● First ATP invested
● Glucose (6C atoms) + ATP, catalysed by Hexokinase → Glucose 6-phosphate +
ADP + H+
,Zooming into ATP = Carrier of Gibbs energy
● 2 high energy phosphate bonds
● Cleaves off one of the phosphate bond → releasing lots of energy
● If enzyme manages to bind this phosphate to the glucose, then that's where the energy
remains as an investment to trigger the start of the pathway
● AMP: last phosphate is not a high energy bond
● So only ADP is what we’re interested in
Gibbs energy is the driving force of all biological processes
● Second Law of Thermodynamics: In all spontaneous processes Gibbs energy is
dissipated (=disappearing)… at constant (environmental) temperature and pressure
○ Gibbs energy not exactly the same as normal energy
○ If process happens and the heat released remains in cell, then this law doesn’t
apply
○ In principle, we are open system, so we constantly exchange heat with our
environment
○ Gas takes up place and volume
○ We assume that this is always true
○ Example: glucose molecules go through the selectively permeable membrane
from high concentration to low concentration (by glucose transporters). In
principle they can also go in the other direction. Reversible process. The probability of glucose going from
left to right is higher than right to left.
■ Entropy: probability creates a net flow from left to right
○ Example 2: sodium (Na+ ions). Selectively permeable membrane has a membrane potential (positive and
negative side). This allows Na+ ions to move to the more negative side. However entropy still allows
probability to create a net flow from left to right (negative side to positive side). Therefore, gibbs energy is
needed. You can't predict the net direction of ion flow if you don't know how to balance these forces
(entropy and energy)
■ Energy: electrical force creates a flow from right to left
Gibbs energy balances energy and entropy
● Change of gibbs energy at constant temperature and pressure, then it is
composed of change in energy (U) plus the pressure times the volume
change minus the temperature (Kelvin) times change in entropy
● This change needs to be smaller than 0 → process then can occur in the
direction that you’ve assigned to
○ If energy is decreasing (negative delta G) → process will occur
○ If entropy change is positive → net effect is that there will be negative contribution to delta G
● If it is bigger than 0, the reverse will happen (the opposite process will have negative delta G)
● If there is a chemical equilibrium, then the Na+ ions for instance will be equal to each other, delta G will be 0.
In a nutshell:
● If ΔG is negative (ΔG < 0) → reaction releases energy, so it's spontaneous.
● If ΔG is positive (ΔG > 0) → reaction requires energy, so it's non-spontaneous (needs help).
● If ΔG is zero → reaction is at equilibrium (goes forward and backward equally).
ATP hydrolysis drives biochemical reactions
● ATP splits off a phosphate to produce ADP + inorganic
phosphate = hydrolysis
● Hydrolysis = breaking a bond by the addition of a H2O
molecule
● ATP + H2O → ADP + Inorganic phosphate
● This hydrolysis reaction has a very negative delta G → driving force for many processes
, ● Driving force of ATP hydrolysis
a. Negative charges on phosphate repel each other → drives hydrolysis reaction in a forward direction
b. Resonance stabilisation of inorganic phosphate (Pi)
i. Phosphate molecule has 4 Oxygen around it
ii. Phosphate molecule exists in many states
iii. Many ways the double bond can be spread around
the molecule
iv. Resonance structures → resonance stabilization
v. Entropy force driving the reaction in the forward direction
c. 2 molecules are formed from 1
d. ADP and inorganic phosphate (Pi) are stabilized by bound water molecules
● However, it is difficult (and impractical) to calculate ΔG from the laws of physics
● We need the gibbs energy bc the driving force is not just energy but also entropy
Reaction gibbs energy is measured
A+B→C+D
● ΔG0’ (delta G 0 prime) is the standard Gibbs energy under biological standard
conditions:
○ pH = 7.0, concentrations 1 M, partial pressure of gases 1 atm
○ Meaning: it is not just the conc of 1 M of each of the molecules, but
also the partial pressure of gases at 1 atm. pH 7 means that protons
aren’t part of the calculations
● Terminology: Gibbs energy = free energy = Gibbs free energy
● Table:
○ ATP has a very strongly negative value
○ G6P hydrolysis is also negative (fact check this in book)
○ Adding phosphate, it would be the same number but positive
Computing reaction gibbs energy
● In cell we don't have every compound at the concentration of 1 molar
otherwise cell would explode, in reality, it is in many order of magnitude
smaller
● Therefore, the real change of gibbs energy in the cell is the standard form
(received from database) plus the gas constant * the absolute temperature * logarithm
of the product concentration divided by substrate concentration
● ΔG0’ can be related to the equilibrium constant of the reaction (see the eq to the right)
● You can derive this equation, because at equilibrium: ΔG = 0 AND [C][D]/[A][B] = Keq … or memorize it
● At equilibrium, the ratio of product and substrate would be equal to the equilibrium constant (which can be any
number !!!!) it is a special property of that reaction
● You let the reaction run until nothing changes, measure the conc,
then you’ll have the equilibrium constant, fill in this equation and
you’ll have the standard gibbs energy
ΔG0’ is additive
Hexokinase reaction
● 2 reactions
○ Hydrolysis of atp
○ Inverse reaction of hydrolysis of G6P (so instead of
hydrolysing G6P, you make it). It’s the same reaction but just
in the other direction → take minus of the delta G
● Total reaction of hexokinase is the addition of the 2 values of delta G
L1 – ATP & Gibbs Free Energy – Enzyme Kinetics
Learning objectives
● How is ATP made? How does the cell know how much to make?
● Proteins consist of amino acids, but how are they made from sugar and other
nutrients?
● How does the cell synthesize the building blocks of cells?
Metabolism
● Is relevant for several diseases eg cancer, diabetes, metabolic syndrome
● is key for bio-based economy
Basic principles
● Metabolism is constrained by the laws of chemistry & physics → energy: the laws
of thermodynamics
● In healthy metabolic network, supply and demand of nutrients are balanced to
support growth or other vital functions → kinetics & regulation
● We take a quantitative approach, but physical and chemical principles should
always serve to understand biological function
Glucose: favourite fuel
● Brain and tumor cells particularly love glucose as a carb source
● Holds for many microbes as well, not all
● Other fuels for energy are used eg fats (long term storage: fasting)
● Proteins (muscles) can also be degraded to amino acids → another source for fuel to get ATP (unhealthy)
● Glycolysis: conversion of glucose → pyruvate (3C atoms, not yet CO2, not fully oxidised to glucose)
● Glycolysis in movement science:
○ Short and intense exercise depends primarily on glycolysis
○ Glycolysis can be upregulated 400x during a 100m sprint
● Glycolysis in cancer research
○ Diagnosis: radioactive analogue of glucose → PET scan shows highly glycolytic tumor lesions
○ Treating tumors: giving inhibitors of glycolytic enzymes & pathways, however targeting is tricky
(normal cells can be affected)
Glycolysis overview – details in tomorrow’s lecture
● To know: substrates of each reaction and the enzyme catalysing it
● No need to know the structures of the chemical compounds, but helpful to look at
● We start with a sugar with 6 Carbon atoms
● Stage 2: aldolase splits the compound into 2 compounds with each 3 Carbon atoms
○ DHAP and GA3P = can be interconverted very easily, reversible process, each can
be converted into pyruvate
○ 1 glucose → 2 pyruvate
● Glycolysis is the first phase of glucose catabolism
● ATP is a carrier of Gibbs energy (needed to start glycolysis)
○ Investment: 2 ATP
○ Gross yield: 4 ATP
○ Net yield: 2 ATP
● In stage 1: 2 molecules of ATP invested
● In stage 3: is done twice, therefore 2 X 2ATP produced
● Microorganisms have many variants of the canonical Embden-Meyerhof-Parnas pathway
(=glycolysis)
,Electron carriers
Biochemical redox reactions involve a limited number of electron
carriers. Reactive sites: where electrons are taken up. Conjugated
systems of alternative double bond and single bond → easily
shuffle around electrons. A few examples:
1. NADH is an electron carrier
● Many redox reactions in cells (electrons transferred
from 1 molecule to another)
● Often these reactions don't directly transfer electrons
to final acceptor
● As a stepping stone, they use NAD to store the electrons for a while → it makes it a universal currency of
electrons in the cell, just like ATP is a universal currency of energy
● If one reaction has excess electrons, it can just drop electron to NAD to produce NADH → NADH can then give
electrons wherever needed in the cell
2. NADP+
3. FAD
2 electrons come with a proton
● Electrons don't come alone
● They come with a H+ that then form a bond
● Carbon has 4 electron pairs (8 electrons) around it
● In these aromatic rings, the double bonds can just shift around the electrons and the
double bond goes to the Nitrogen (previously positively charged) → neutralised
NADH
● Large molecules like NADH needed to facilitate these biological processes because it
can absorb electrons and give them away easily
Alcoholic fermentation
● The formation of ethanol re-oxidizes NADH without an external electron acceptor → Fermentation
● Examples: yeast, goldfish, rice root.
● Some yeasts also ferment in the presence of O2, when glucose is abundant
The fate of pyruvate
● Lactate and ethanol are fermentation products.
● Fermentation is the process in which NADH generated in glycolysis donates its electrons to an internal, organic
electron acceptor (pyruvate).
● Under anaerobic conditions, fermentation is necessary to generate ATP.
● So pyruvate has 3 fates
a. Acetyl coa → Further oxidation
b. Fermentation → Lactate: in muscles or lactic acid bacteria
c. Fermentation → Ethanol
i. Fermentation: No o2, you need internal acceptor of electron = balance
of synthesis
Enzymes of glycolysis
1. Hexokinase
● Making a phosphate bond is energetically costly
● First ATP invested
● Glucose (6C atoms) + ATP, catalysed by Hexokinase → Glucose 6-phosphate +
ADP + H+
,Zooming into ATP = Carrier of Gibbs energy
● 2 high energy phosphate bonds
● Cleaves off one of the phosphate bond → releasing lots of energy
● If enzyme manages to bind this phosphate to the glucose, then that's where the energy
remains as an investment to trigger the start of the pathway
● AMP: last phosphate is not a high energy bond
● So only ADP is what we’re interested in
Gibbs energy is the driving force of all biological processes
● Second Law of Thermodynamics: In all spontaneous processes Gibbs energy is
dissipated (=disappearing)… at constant (environmental) temperature and pressure
○ Gibbs energy not exactly the same as normal energy
○ If process happens and the heat released remains in cell, then this law doesn’t
apply
○ In principle, we are open system, so we constantly exchange heat with our
environment
○ Gas takes up place and volume
○ We assume that this is always true
○ Example: glucose molecules go through the selectively permeable membrane
from high concentration to low concentration (by glucose transporters). In
principle they can also go in the other direction. Reversible process. The probability of glucose going from
left to right is higher than right to left.
■ Entropy: probability creates a net flow from left to right
○ Example 2: sodium (Na+ ions). Selectively permeable membrane has a membrane potential (positive and
negative side). This allows Na+ ions to move to the more negative side. However entropy still allows
probability to create a net flow from left to right (negative side to positive side). Therefore, gibbs energy is
needed. You can't predict the net direction of ion flow if you don't know how to balance these forces
(entropy and energy)
■ Energy: electrical force creates a flow from right to left
Gibbs energy balances energy and entropy
● Change of gibbs energy at constant temperature and pressure, then it is
composed of change in energy (U) plus the pressure times the volume
change minus the temperature (Kelvin) times change in entropy
● This change needs to be smaller than 0 → process then can occur in the
direction that you’ve assigned to
○ If energy is decreasing (negative delta G) → process will occur
○ If entropy change is positive → net effect is that there will be negative contribution to delta G
● If it is bigger than 0, the reverse will happen (the opposite process will have negative delta G)
● If there is a chemical equilibrium, then the Na+ ions for instance will be equal to each other, delta G will be 0.
In a nutshell:
● If ΔG is negative (ΔG < 0) → reaction releases energy, so it's spontaneous.
● If ΔG is positive (ΔG > 0) → reaction requires energy, so it's non-spontaneous (needs help).
● If ΔG is zero → reaction is at equilibrium (goes forward and backward equally).
ATP hydrolysis drives biochemical reactions
● ATP splits off a phosphate to produce ADP + inorganic
phosphate = hydrolysis
● Hydrolysis = breaking a bond by the addition of a H2O
molecule
● ATP + H2O → ADP + Inorganic phosphate
● This hydrolysis reaction has a very negative delta G → driving force for many processes
, ● Driving force of ATP hydrolysis
a. Negative charges on phosphate repel each other → drives hydrolysis reaction in a forward direction
b. Resonance stabilisation of inorganic phosphate (Pi)
i. Phosphate molecule has 4 Oxygen around it
ii. Phosphate molecule exists in many states
iii. Many ways the double bond can be spread around
the molecule
iv. Resonance structures → resonance stabilization
v. Entropy force driving the reaction in the forward direction
c. 2 molecules are formed from 1
d. ADP and inorganic phosphate (Pi) are stabilized by bound water molecules
● However, it is difficult (and impractical) to calculate ΔG from the laws of physics
● We need the gibbs energy bc the driving force is not just energy but also entropy
Reaction gibbs energy is measured
A+B→C+D
● ΔG0’ (delta G 0 prime) is the standard Gibbs energy under biological standard
conditions:
○ pH = 7.0, concentrations 1 M, partial pressure of gases 1 atm
○ Meaning: it is not just the conc of 1 M of each of the molecules, but
also the partial pressure of gases at 1 atm. pH 7 means that protons
aren’t part of the calculations
● Terminology: Gibbs energy = free energy = Gibbs free energy
● Table:
○ ATP has a very strongly negative value
○ G6P hydrolysis is also negative (fact check this in book)
○ Adding phosphate, it would be the same number but positive
Computing reaction gibbs energy
● In cell we don't have every compound at the concentration of 1 molar
otherwise cell would explode, in reality, it is in many order of magnitude
smaller
● Therefore, the real change of gibbs energy in the cell is the standard form
(received from database) plus the gas constant * the absolute temperature * logarithm
of the product concentration divided by substrate concentration
● ΔG0’ can be related to the equilibrium constant of the reaction (see the eq to the right)
● You can derive this equation, because at equilibrium: ΔG = 0 AND [C][D]/[A][B] = Keq … or memorize it
● At equilibrium, the ratio of product and substrate would be equal to the equilibrium constant (which can be any
number !!!!) it is a special property of that reaction
● You let the reaction run until nothing changes, measure the conc,
then you’ll have the equilibrium constant, fill in this equation and
you’ll have the standard gibbs energy
ΔG0’ is additive
Hexokinase reaction
● 2 reactions
○ Hydrolysis of atp
○ Inverse reaction of hydrolysis of G6P (so instead of
hydrolysing G6P, you make it). It’s the same reaction but just
in the other direction → take minus of the delta G
● Total reaction of hexokinase is the addition of the 2 values of delta G
