index
Announcements
Questions
Section 10.4: Compartmentation and Metabolism
Section 10.5: Thermodynamics of Metabolic Reactions
Coupling of Unfavorable and Favorable Reactions
Section 10.6: The Free Energy of ATP
Structural Basis of Energy in ATP
Hierarchy of Energy Carriers
Section 10.7: The Metabolic Roles of ATP
Phosphoryl Group Transfer in Glutamate Synthesis
Phosphagens are High Energy Phosphoamides
Section 10.8: Free Energy of Thioesters
Section 10.9: Redox Potentials
Oxidation-Reduction Reactions
Measurement of Redox Potentials
Measurement of Redox Potentials, 2
Standard Reduction Potentials
Redox Potentials and Free Energy
Example: Free Energy Change for Reduction of O₂
Section 10.10: Experimental Methods
Questions
Next Lecture: Sections 11.1 - 11.2

Fundamentals of Biochemistry

Biochem 380 - Fall 2006

Lecture 030

Outline

Announcements
Questions from previous lecture
Section 10.4: Compartmentation and Metabolism
Section 10.5: Thermodynamics of Metabolic Reactions
Section 10.6: The Free Energy of ATP
Section 10.7: The Metabolic Roles of ATP
Section 10.8: Free Energy of Thioesters
Section 10.9: Redox Potentials
Section 10.10: Experimental Methods

Announcements

Lecture 29 notes are online
The answer key and scores for quiz 006 have been uploaded to the course web page
Lecture schedule and exam coverage have been revised:
- Exam II will cover chapters 6 - 11
- Chapter 21 (Transcription) has been dropped from the schedule
Homework problems for Chapter 10:
- 2, 3, 7, 8, 14

Questions

Any questions on the material from the previous lecture?

Section 10.4: Compartmentation and Metabolism

Compartmentation is an important aspect for controlling metabolism. It refers to the physical separation of different metabolic processes
Physical separation can occur in a variety of ways. At the molecular level, multi-enzyme complexes can operate on a local pool of metabolites. At the intracellular level, membrane-bound organelles can segregate different pathways. At the multicellular level, different cell types can be engaged in specialized metabolic activities
Compartmentation allows for opposing metabolic pathways to operate simultaneously. Examples include eukaryotic cells, where fatty acid synthesis occurs in the cytosol and fatty acid breakdown occurs in the mitochondria. At the multicellular level, heterocysts are cells in Anabaena spherica that do nitrogen fixation in a low-oxygen environment, and so must be segregated from photosynthetic cells that produce oxygen

Section 10.5: Thermodynamics of Metabolic Reactions

Previously, we have learned that for any particular reaction to be spontaneous or thermodynamically favorable, the reaction must have a negative change in free energy
Likewise, for a sequence of reactions in a metabolic pathway, the overall change in free energy for the entire sequence must also be negative
The overall free energy, ΔG, is a function of the standard free energy and concentration:

ΔG = ΔG°' + RT ln [C][D] / [A][B]

In cells, the actual free energy can be made favorable through control of metabolite concentrations. This can be done using compartmentation, as well as by controlling relative reaction rates at different steps in a pathway
Another way for producing an overall negative ΔG for a reaction is to couple an unfavorable reaction to a favorable one, such as the hydrolysis of ATP

Coupling of Unfavorable and Favorable Reactions

An example of changing the standard free energy of a reaction by coupling it to another one can be seen below:

A → B + C	ΔG°' = +21 kJ/mol
B → D	ΔG°' = -34 kJ/mol
______________________________
A → C + D	ΔG°' = -13 kJ/mol

In this example, the second reaction involving B and D can represent a favorable reaction such as the hydyrolysis of ATP
The coupling of the second reaction to the first can be done in a number of ways
One way might involve the conformational change in an enzyme-substrate complex upon hydrolysis of ATP, where the energy of hydrolysis is used to create a geometrically-strained, 'reactive' conformation
Other examples of energy coupling include the formation of complexes that are chemically unstable

Section 10.6: The Free Energy of ATP

As we have heard probably a 1,000,000 times so far, ATP is the main energy carrier molecule in cellular metabolism
Its solubility, small size and specific shape enable it to be used as a specific substrate for a large number of nucleotide-binding enzymes
In addition, its hydrolysis to ADP and Pi produces a signficant amount of free energy that is available for coupling to less favorable reactions
The standard free energy change of hydrolysis is around -30 kJ/mol, and under physiological conditions where there is a higher concentration of ATP, the overall free energy change is closer to -50 kJ/mol

Structural Basis of Energy in ATP

Three factors contribute to the amount of energy that is obtained in the hydrolysis of ATP:
- Resonance stabilization
- Electrostatic repulsion
- Hydration

Four different resonance structures are possible in the orthophosphate product of the reaction:

The separation of the 4 negative charges results in greater electrostatic stabilization and
The increase in solvent exposure increases the stablization due to hydration

Hierarchy of Energy Carriers

Although the free energy change of ATP is rather large, it sits somewhere in the middle of the hierarchy of energy carrier molecules
The following table gives a comparison of free energies for a number of phosphorylated compounds:

The intermediate position of ATP allows it to be efficiently coupled to other reactions of greater and lesser energy change, somewhat analogous to the voltage level of household electricity, which is intermediate in value between power sources and power-consuming devices

Section 10.7: The Metabolic Roles of ATP

How exactly is the large negative energy of ATP hydrolysis coupled to less favorable reactions in metabolism? Part of the coupling can be understood by considering the potential energy associated with the transferred phosphoryl group
Such a transfer can activate a substrate, by creating a high-energy state that can undergo a reaction to a more stable state, with a negative ΔG for the reaction, compared to one which would have a positive ΔG if the substrate did not have the phosphoryl group
We'll look at an example of this in a reaction for glutamine synthesis from glutamate
In general, the ability of a phosphoryl group to be transferred from one compound to another can be quantified as the phosphoryl-group-transfer potential. ATP itself can be produced from other compounds that have a higher transfer potential
Some of these high-potential compounds, such as phosphoenolpyruvate, are seen in glycolysis. Others, such as the phosphagens (phosphocreatine, phosphoarginine) are used for energy storage

Phosphoryl Group Transfer in Glutamate Synthesis

An example of the mechanism of energy coupling is seen in the synthesis of glutamine from glutamate. The enzyme glutamine synthetase catalyzes the production of a γ-glutamyl phosphate intermediate
This is an energy-rich intermediate that is unstable in water, but is protected in the active site of the enzyme. The resulting phosphate is a good leaving group, which can be displaced by the ammonia nucleophile to form glutamine

Phosphagens are High Energy Phosphoamides

Phosphagens are energy-rich phosphate storage molecules found in animal muscle cells. They include phosphocreatine (found in vertebrates) and phosphoarginine (invertebrates)
These are phosphoamides with a higher transfer potential than the phosphoanhydride linkages in ATP. Phosphocreatine is a significant source of energy in resting muscle cells in animals, with a concentration about 5 times greater than ATP
It can provide 3 to 4 seconds bursts of energy by being converted to ATP by creatine kinase:

Phosphocreatine + ADP → Creatine + ATP

Section 10.8: Free Energy of Thioesters

Thioesters are another type of compound with high transfer potentials. One example that we have already seen is the thioester linkage in coenzyme A, where it is joined to an acetyl group
The energy associated with the thioester linkage is similar to that of the phosphoanhydride linkage in ATP. For example, the standard energy of hydrolysis for acetyl CoA is -31 kJ/mol
In addition to carbon transfer, the high energy of a CoA thioester is used to generate ATP (or GTP) in the fifth step of the citric acid cycle:

Section 10.9: Redox Potentials

The potential for electrons to be transferred in oxidation-reduction reactions is measured by E_o', the standard reduction potential
The standard reduction potential enables a quantitative evaluation of the energy changes that occur during oxidation-reduction reactions
We'll cover the following items related to the standard reduction potential:
- Definitions and terms used in oxidation-reduction reactions
- Experimental determination of the standard reduction potential
- The relation between ΔE_o' and the standard free energy change ΔG°'
- Sample calculations of ΔG°' using ΔE_o'

Oxidation-Reduction Reactions

Before considering redox potentials, some definition of terms will be helpful
For a given substance that can exist in the oxidized and reduced states, let X^- refer to the reduced state and X refer to the oxidized state
Here, X and X^- refer to a redox couple and the half-reaction for the couple is:

X^- → X + e^-

Additionally, X^- can be referred to as the reductant and X can be referred to as the oxidant
For a complete oxidation-reduction reaction, two redox couples are involved, with one species as the donor D in the reduced state and the other species as the acceptor A in the oxidized state:

D^- + A → D + A^-

This full reaction corresponds to the following two half-reactions:

D^- → D + e^-

A + e^- → A^-

Measurement of Redox Potentials

The reduction potential of a species X to go from the reduced state to the oxidized state can be quantified by measuring the potential for electron flow when the redox couple of the species is electrically connected to a reference redox couple, (H⁺:H₂)
In the apparatus above, the redox couple for X is placed in the sample cell on the left and the reference couple in the cell on the right, with both couples at 1M concentration at equilibrium
A voltage meter is connected in series between the two solutions and an agar bridge completes the electrical circuit

Measurement of Redox Potentials

If under these initial conditions the current flows to the right, we can conclude that the following half-reactions are occurring in the cells:

X^- → X + e^-

H⁺ + e^- → 1/2 H₂

This means that the complete reaction X^- + H → X + 1/2 H₂ proceeds in the forward direction, and so the reduction potential of the couple (X:X^-) is negative with respect to (H⁺:H₂)
Conversely, if the current flowed to the left, then the reduction potential of the couple would be positive

Standard Reduction Potentials

Below is a table of some standard reduction potentials (measured at pH 7 instead of pH 0):

Redox Potentials and Free Energy

For any given oxidation-reduction reaction, each redox couple of the reaction will have a standard reduction potential
The difference in reduction potentials between the two couples, ΔE_o', will reflect the direction that the reaction will proceed, where ΔE_o' is defined as:

ΔE_o' = E_o'_{electron acceptor} - E_o'_{electron donor}

A relation exists between the change in standard reduction potential ΔE_o' and the standard free energy change ΔG°':

ΔG°' = -n F ΔE_o'

n = number of electrons
F = the Faraday constant, 96.48 kJ/mol V

Example: Free Energy Change for Reduction of O₂

As an example, consider the reduction of O₂ by NADH:

1/2 O₂ + NADH + H⁺ → H₂O + NAD⁺

This can be rewritten as a sum of the following two half-reactions from Table 10.4:

NAD⁺ + 2H⁺ + 2 e^- → NADH + H⁺		E_o' = -0.32 V
1/2 O₂ + 2 H⁺ + 2 e^- → H₂O		E_o' = +0.82 V

The net change in the standard reduction potential is ΔE_o' = 0.82 V - (-0.32 V) = 1.14 V
Then the change in standard free energy is:

ΔG°' = -(2)(96.48 kJ/mol V)(1.14 V) = -220 kJ/mol

This is a large amount of energy. By comparison, the hydrolysis of ATP is -31.4 kJ/mol, so the maximum free energy obtained per oxidation of a single NADH is equivalent to about 7 ATP

Section 10.10: Experimental Methods

Experimental investigations of metabolism are very challenging because of the complexity of the many pathways involved. Many experimental approaches have been developed over the years to characterize the enzymes, intermediates, flux and regulation in these pathways
One very powerful approach makes use of radioactive isotopes such as ³H (tritium) and ¹⁴C. These can be selectively added to a substrate of interest, allowing tracing of its fate through a pathway of reactions
When an enzyme or metabolite has been identified, it can then be isolated in an in vitro system, where its properties can be investigated under known, controlled conditions. For enzymes, such properties include determination of substrate specificity and kinetic parameters, as well as susceptibility to various inhibitors
Another valuable technique involves the creation of mutant organisms in order to identify genes that are associated with particular nutrient requirements. For example, genes necessary for the synthesis of certain amino acids can be identified from mutant strains of bacteria which only grow on an external medium that supplies the missing amino acid

Questions

Questions about the material covered today?

Next Lecture: Sections 11.1 - 11.2

Read Sections 11.1 - 11.2

Table of Contents

Fundamentals of Biochemistry

Biochem 380 - Fall 2006

Lecture 030

Outline

Announcements

Questions

Section 10.4: Compartmentation and Metabolism

Section 10.5: Thermodynamics of Metabolic Reactions

Coupling of Unfavorable and Favorable Reactions

Section 10.6: The Free Energy of ATP

Structural Basis of Energy in ATP

Hierarchy of Energy Carriers

Section 10.7: The Metabolic Roles of ATP

Phosphoryl Group Transfer in Glutamate Synthesis

Phosphagens are High Energy Phosphoamides

Section 10.8: Free Energy of Thioesters

Section 10.9: Redox Potentials

Oxidation-Reduction Reactions

Measurement of Redox Potentials

Measurement of Redox Potentials

Standard Reduction Potentials

Redox Potentials and Free Energy

Example: Free Energy Change for Reduction of O2

Section 10.10: Experimental Methods

Questions

Next Lecture: Sections 11.1 - 11.2

Example: Free Energy Change for Reduction of O₂