Introduction to Aerobic Metabolism

The purpose of this lecture is to provide a general introduction to metabolic principles as well as briefly discuss oxidative stress that results from the use of oxygen as the final electron acceptor in aerobic catabolism.

1) We will briefly discuss an overview of catabolic and anabolic pathways.

2) We will discuss, again in brief, ATP as the universal currency of metabolic energy and also review vitamins that are precursors for many of the coenzymes used in metabolism.

3) The use of oxygen as the terminal electron acceptor in the oxidation of foodstuffs permits much greater energy to be obtained from their breakdown than would be possible in its absence. However, a byproduct of this role for oxygen is the formation of toxic free radicals that result from incomplete reduction of oxygen. We will discuss antioxidant defenses used to reduce toxic free radicals in the cell.

At first glance, metabolism appears to be a bewildering array of chemical reactions. The figure to the right represents over 500 different chemical intermediates and a greater number of enzymes. Virtually all organisms carry out the same basic set of metabolic pathways. Despite these large number of enzyme mediated reactions, they actually represent a highly integrated, and tightly regulated, process. We will only cover a very small subset of these reactions in the course.

1. Overview of metabolic pathways

From Garrett & Grisham “Biochemistry”

A more useful way to illustrate an overview of metabolism is shown to the right with pathways color-coded. This provides a first glimpse of interrelationships between major pathways. For instance carbohydrate, lipid and some amino acid metabolism converge at the formation of Acetyl CoA (dark circle) prior to entering the Krebs cycle (blue circle at the lower center of the diagram.) Probably most important, and placed centrally in this diagram, is carbohydrate metabolism, which will be discussed in detail in the next three lectures.

From Berg, Tymoczko & Stryer “Biochemistry, 5th ed”

Metabolism can be divided into two major processes, termed catabolism and anabolism.

Catabolism is the process of breaking down the larger, reduced, compounds such as glucose, amino acids or fatty acids. Energy is released as electrons are transferred from these reduced compounds ultimately to oxygen forming to small end products such as CO2, H2O and NH3 to yield energy. These processes are oxidative (meaning substrates are loosing electrons) and exergonic (energy releasing). In order to capture the energy from these compounds, the oxidation reactions must be coupled with reactions that can store the energy chemically, otherwise the energy will be released as heat. The many steps often used in catabolic processes allow smaller packets of energy to be released and stored chemically, usually ultimately as ATP.

Anabolism is the reverse process of building the macromolecules that run the processes of the cell. Driving the reductive, endergonic, reactions is the chemical energy largely stored in the form of ATP and reduced NADPH. NADPH is used almost exclusively for reductive biosynthesis, while NADH is used in the generation of ATP as will be discussed in the next few lectures. Whereas catabolic pathways converge to only a very few end products, anabolic pathways diverge to synthesize a huge variety of molecules from a small set of building blocks. From Garrett & Grisham “Biochemistry”

Overview of catabolism.

It is useful to classify the process of catabolism as comprising three stages. The first stage involves the breakdown of large macromolecules into their component building blocks. The second stage involves the degradation of these building blocks into a common product, Acetyl CoA (with some products of amino acid degradation producing intermediates in the citric acid cycle). The final stage of catabolism is the aerobic combustion of the acetyl groups of acetyl CoA by the citric acid cycle and oxidative phosphorylation to produce CO2 and H20. As will be discussed in lecture 27, oxidation of acetyl CoA generates most of the energy produced by the cell. From Garrett & Grisham “Biochemistry”

A central molecule in metabolism, about which you will hear much more in the next few lectures, is Acetyl CoA. This molecule is an activated carrier of two carbon units, acquiring them in the oxidation of carbohydrate, fats and amino acids. Shown below is the structure of Coenzyme A. The sulfhydryl group (left) can be linked with acyl groups by thioester bonds. Coenzyme A is formed from ADP (with a phosphorylated 3’ hydroxyl on the ribose sugar), plus pantothenate (a B vitamin) and a -mercaptoethylamin unit. We will briefly discuss vitamins slightly later in the lecture.

Coenzyme A:

From Berg, Tymoczko & Stryer “Biochemistry, 5th ed”

2. ATP, energy, coenzymes and vitamins

Life requires the continual input of energy for building macromolecules, maintaining ionic gradients, motion and many other purposes. ATP is considered the universal currency of free energy in biological systems. Hydrolysis of ATP to ADP (or AMP) is a highly favorable reaction (Gº = -7.3 kcal/mol) due to the resonance bonding and electrostatic repulsion of phosphates. By coupling its hydrolysis with otherwise unfavorable reactions it can drive such reactions that are key to living processes (see below). ATP is continually being used in energy requiring processes and regenerated during catabolism through processes discussed in the next three lectures.

Favorable process (-G )

Unfavorable process (+G)

Coupling of a Favorable process (-G ) with an Unfavorable (+G ) yields an overall favorable reaction (-G )

G G

ATP is the universal currency of metabolic energy, but it is constantly being spent and regenerated. It is estimated that ATP stores (~4mM) provide sufficient energy to maintain muscle contraction for only a second. Muscle also contains creatine phosphate (~25mM) that can be used to generate ATP and thus supply energy for a few seconds. (Creatine phosphate is the major source for generating ATP during the first 4 seconds of a 100 meter sprint.) After these stores are used up, ATP must be generated through metabolism. As will be discussed in Block III, glycogen provides a readily mobilized storage of glucose, particularly in the muscle and liver. Glucose units from glycogen can be degraded through glycolysis anaerobically to yield a small quantity of ATP or aerobically to produce much more ATP if oxygen is sufficient.

From Berg, Tymoczko & Stryer “Biochemistry, 5th ed”

Carbon Fuels: The transient stores of ATP are constantly being replenished by the oxidation of carbon fuels. Shown below are the free energies associated with oxidation of one-carbon compounds to carbon dioxide. Physiological fuels are obviously more complex (bottom), but the trends in free energy of oxidation will be similar.

Glucose is an extremely important fuel that is universally and, under well-fed circumstances, the sole energy source for the brain. However, fats provide a more efficient long-term energy storage since their carbon atoms are more reduced than those of sugars.

Thiamine (B1) Thiamine pyrophosphate Aldehyde transfer

Riboflavin (B2) Flavin adenine dinucleotide (FAD) Oxidation-reduction

Pyridoxine (B6) Pyridoxal phosphateGroup transfer to or from amino acids

Nicotinic acid (niacin- B3)

Nicotinamide adenine dinucleotide (NAD+) Oxidation-reduction

Pantothenate (B5) Coenzyme A Acyl-group transfer

Biotin Biotin-lysine complexes (biocytin)ATP-dependent carboxylation and carboxyl-group transfer

Folic acid TetrahydrofolateTransfer of one-carbon components; thymine synthesis

B12 5’ Deoxyadenosyl cobalaminTransfer of methyl groups; intramolecular rearrangements

C (ascorbic acid) Antioxidant

Vitamin Coenzyme Typical reaction type

Vitamins are small organic molecules required in small amounts in the diet of higher animals, several of which are used as precursors to coenzymes, as has already been alluded to earlier in the course. You will be exposed to vitamins at various points throughout the course; this chart is to provide some familiarity with them. Water-Soluble Vitamins

Beriberi

Cheliosis, dermititis

Depression, convulsions

Pellagra

Hypertension

Rash, muscle pain

Anemia, neural-tube defects in development

Anemia

Scurvy

Consequences of deficiency

The B-vitamins are components of coenzymes.

Pyridoxal PhosphateInvolved in transferring amino groups

Coenzyme A

(Coenzyme A)

(FAD)

(NAD+)

From Berg, Tymoczko & Stryer “Biochemistry, 5th ed”

Adenosine diphosphate (ADP) is a fundamental building block in key metabolic compounds including ATP, NADH, FAD and Coenzyme A. This suggests ADP is an ancient module in the evolution of metabolic pathways.

From Berg, Tymoczko & Stryer “Biochemistry, 5th ed”

ARoles in vision, growth, reproduction

Night blindness, cornea damage, damage to respiratory and gastrointestinal tract

DRegulation of calcium and phosphate metabolism

Rickets (children): skeletal deformaties, impaired growth

Osteomalacia (adults): soft, bending bones

E AntioxidantInhibition of sperm production; lesions in muscles and nerves (rare)

K Blood coagulation Subdermal hemorrhaging

Fat-Soluble Vitamins

Vitamin Function Consequences of Deficiency

A hallmark of eukaryotes is the compartmentalization of different tasks. Metabolism is no exception to such compartmentalization. Many tasks involve shuttling components between the cytosol and mitochondria. As an example, consider glucose metabolism below. Glycolysis takes place in the cytosol. To continue the breakdown of pyruvate under aerobic conditions, it is transported into the mitochondria, which contains the enzymes of the citric acid cycle and the enzymes and membrane structure used for oxidative phosphorylation and electron transport.

From Garrett & Grisham “Biochemistry”

Aerobic catabolism permits much greater energy from the breakdown of glucose (and other compounds) than is possible under anaerobic conditions. This high yield, however, comes at the price of potential poisoning by intermediates in the reduction of oxygen. These intermediates are termed reactive oxygen species (ROS). Although most of the oxygen consumed during aerobic metabolism is fully reduced to H20, some free radicals are produced as byproducts. In addition, reactions of oxygen with drugs and environmental toxins can also add to the level ROS. It is estimated that 3 to 5% of the oxygen we consume is converted to oxygen free radicals. Cells contain antioxidant enzymes designed to remove ROS. When the ability of the cell to deal with these ROS is overwhelmed, oxidative stress results.

3. Oxidative Stress

From “Marks – Basic Medical Biochemistry, a Clinical Approach”

Oxygen radicals, like all radicals, have a single unpaired electron in an orbital. (A free radical is one that is capable of independent existence.) Radicals are highly reactive because they will extract an electron from a neighboring molecule to fill their orbital, which can initiate a chain reaction. The superoxide anion, resulting from a single electron transfer to oxygen, will not diffuse far from the site of origin, but can generate other radicals. Hydrogen peroxide is not a radical, but can participate in the generation of free radicals (see right) and can diffuse through membranes to expand the extent of free radical damage. The hydroxyl radical is the most reactive species and can be produced from hydrogen peroxide and superoxide, through reactions shown to the right. Oxidative stress is thought to contribute to a large number of disease states (see below).

Some disease states associated with oxidative stress

From “Marks – Basic Medical Biochemistry, a Clinical Approach”

Superoxide dismutase converts superoxide to molecular oxygen and hydrogen peroxide in two steps.

The first line of cellular defense against oxygen radicals is the enzyme superoxide dismutase (SOD). There are three important isoforms of SOD, including a Cu/Zn protein in the cytosol, a Mn protein in the mitochondria and an extracellular Cu/Zn enzyme. SOD catalyzes the reaction of 2 superoxide molecules (O2

-) to form one molecule of O2 and one of hydrogen peroxide (H2O2). Catalase is a key enzyme that rids the cell of hydrogen peroxide and is found largely in peroxisomes (organelles that generate a fair bit of hydrogen peroxide during long-chain fatty acid oxidation and other reactions), with smaller amounts in the cytosol.

From “Marks – Basic Medical Biochemistry, a Clinical Approach”

A third important mechanism for the cell’s defense of oxidative stress is the use of reduced glutathione (G-SH) to detoxify hydrogen peroxide (see right). Glutathione peroxidase catalyzes the reaction of reduced glutathione (G-SH) and hydrogen peroxide to form oxidized glutathione (G-S-S-G) and water. Reduced glutathione is then regenerated by glutathione reductase, using electrons donated by NADPH.

Fig. 13.6 from Lippincott

A number of exogenous antioxidant compounds are also thought to be able to contribute to the detoxification of reactive oxygen species. Included among these are vitamins C (below) and E (right). Vitamin E may play an especially important role in protecting membranes from lipid peroxidation since it is hydrophobic in nature. Eating foods rich in these antioxidants has been correlated with reduced risk of various diseases associated with oxidative stress. However, clinical trials with dietary supplements have been less convincing.

L-Ascorbate (Vitamin C), in addition to the classical role it plays in formation of hydroxy proline in collagen, can play a more general role in donating single electrons to free radicals or disulfides as it is oxidized to dehydro-L-ascorbic acid. It may also play an important role in regeneration of Vitamin E. From “Marks – Basic Medical Biochemistry, a Clinical Approach”

Vitamin E (-tocopherol) acts to terminate free radical lipid peroxidation by donating single electrons to lipid peroxyl radicals to form the more stable lipid peroxide (LOOH). As a result, -tocopherol is converted to the fully oxidized version. From “Marks – Basic Medical Biochemistry, a Clinical Approach”

Sample questions:

Which of the following compounds contributes to the cell’s defense against oxidative stress?

A) PyridoxineB) Coenzyme AC) Vitamin ED) BiotinE) Vitamin A

Which of the following is NOT considered a reactive oxygen species (ROS)?

A) SuperoxideB) Hydroxyl ionC) Hydrogen peroxideD) Hydroxyl radical

Study Points :

1) Understand that catabolism refers to the oxidative breakdown of reduced compounds such as glucose, amino acids and fatty acids to form small compounds such as CO2, H2O and NH3 with the release of energy.

2) Understand that anabolism is the reductive process of building a wide variety of compounds using chemical energy that is largely stored in the form of ATP and NADPH.

3) Recognize the general role of B-vitamins as components of important coenzymes used in metabolic processes and be able to recognize key vitamins and their role in metabolism.

4) Understand what is meant by “Oxidative Stress”.

5) Understand that reactive oxygen species (ROS) form during the incomplete reduction of oxygen and be able to identify them.

6) Be able to recognize enzymes involved in cellular defense against oxidative stress along with exogenous antioxidant compounds.