CHAPTER 5 MICROBIAL METABOLISM

METABOLIC REACTIONS

Metabolism-sum of all the chemical reactions within a living organism. 2 main types of reactions are involved:

1. Catabolism (catabolic or degradation reactions)-- these break down larger molecules to smaller ones, breaking chemical bonds and releasing energy in the process (exergonic).

2. Anabolism (anabolic or biosynthetic reactions)- these build larger molecules from smaller ones, building chemical bonds and using energy (endergonic). Biosynthesis is the name for building reactions in living organisms.

Catabolic reactions provide the energy to drive the anabolic reactions. This coupling involves the formation of ATP. ATP stores energy from catabolic reactions and transfers it to anabolic reactions.

Catabolic reactions also provide building blocks for anabolic reactions.

An ATP (adenosine triphosphate) molecule consists of an adenine, a ribose and 3 phosphate groups. A large amount of energy is required to form the bond that attaches the third phosphate group. When this bond is broken, all that energy is released at once, and is used to carry out energy-requiring processes of the cell.

ENZYMES

A cell’s metabolic pathways (which chemical reactions occur in that cell) are determined by which enzymes the cell makes, which is determined by the genes the cell possesses.

Chemical reactions occur as ions, atoms, or molecules collide. Activation energy is the amount of energy needed to disrupt the electron configuration of a molecule so that electrons can be rearranged. Conditions in living cells involve temperatures and pressures that are mostly too low for enough of these collisions to occur with enough force to result in reactions at a rate that will maintain life. To increase the speed of reactions in cells, biological catalysts called enzymes are essential. (Catalyst- substance that can speed a chemical reaction without itself being altered).

Enzymes work in 2 major ways:

1. In catabolic reactions, enzymes hold the larger molecule in such a way that chemical bonds are strained and more easily broken.

2. In anabolic reactions, enzymes hold the smaller molecules in proper relation to each other so that bonds can easily form.

Enzymes lower the activation energy (energy required for the reaction to start) without increasing temperature or pressure inside the cell. Facts to remember about enzymes:

1. The action of enzymes is very specific. The substance on which an enzyme acts is called the substrate. An enzyme acts on only one specific substrate and catalyzes only one specific reaction.

2. Enzymes are large protein molecules with characteristic 3-dimensional shapes. The shape allows the enzyme to fit with its substrate and carry out its function.

3. Enzymes are extremely efficient. Under ideal conditions, an enzyme can speed the rate of a reaction up to 10 billion times. Many enzyme molecules can catalyze a large number of reactions per second (average 1 - 10,000 but up to 500,000). The number of reactions per second is the turnover number of the enzyme.

DNA polymerase 15/sec

Lactate dehydrogenase 1000/sec

4. Enzymes are under cellular controls in 2 ways

a. Enzyme synthesis can be turned on or off

b. Activity of enzyme molecules can be controlled.

5. Enzyme names almost all end in -ase. (Some of the first enzymes discovered were not named this way--- pepsin, for example)

6. There are 6 classes of enzymes, according to type of reaction.

CLASS	TYPE OF REACTION CATALYZED	EXAMPLES
OXIDOREDUC-TASE	OXIDATION-REDUCTION REACTIONS	CYTOCHROME OXIDASE, LACTATE DEHYDROGENASE
TRANSFERASE	TRANSFER OF FUNCTIONAL GROUPS SUCH AS AN AMINO GROUP	ACETATE KINASE, ALANINE DEAMINASE
HYDROLASE	HYDROLYSIS (ADDITION OF WATER)	LIPASE, SUCRASE
LYASE	REMOVAL OF GROUPS OF ATOMS WITHOUT HYDROLYSIS	OXALATE DECARBOXYLASE, ISOCITRATE LYASE
ISOMERASE	REARRANGEMENT OF ATOMS WITHIN A MOLECULE	GLUCOSE-PHOSPHATE ISOMERASE
LIGASE	JOINING OF 2 MOLECULES USING ATP ENERGY	DNA LIGASE

ENZYME COMPONENTS

A few enzymes consist only of protein, but most have at least one other component in addition to the protein. The protein portion is called the apoenzyme. Apoenzymes combine with another component, which may be:

Inorganic, such as a metal ion—this is called a cofactor

Organic but not a protein, often a vitamin—this is called a coenzyme

A few enzymes require both a cofactor and coenzyme. Without the required cofactor or coenzyme, the enzyme cannot function.

2 essential coenzymes in cellular metabolism are:

NAD+ ----nicotinamide adenine dinucleotide

NADP+ ---- nicotinamide adenine dinucleotide phosphate

Both are derived from niacin, one of the B vitamins, and both function as electron carriers in metabolic reactions.

Cofactors are usually metal ions. They form a bridge between enzyme and substrate so that they can connect properly.

MECHANISM OF ENZYME ACTION

1. Surface of the enzyme molecule contains a region called the active site. This is the part that “fits” the substrate.

2. A temporary enzyme-substrate complex forms as the substrate fits into the active site.

3. The substrate is changed. This will involve making or breaking of chemical bonds.

4. Changed substrate no longer fits the active site, so it is released.

5. Enzyme molecule is unchanged and can immediately catalyze another reaction.

FACTORS INFLUENCING ENZYME ACTIVITY

1. Temperature---rate increases as temperature increases, up to ideal temperature. Beyond that, rate decreases because excess heat denatures enzymes, causing them to lose their 3-dimensional shape. Denaturation is usually not reversible- cooking egg white is an example. Denaturation can also occur due to excessive exposure to acids, bases, alcohol, heavy metal ions, UV radiation.

2. pH--- most enzymes have an ideal (optimum) pH, often near neutral, where maximum activity occurs. As the pH shifts above or below this ideal, activity decreases. Extremes cause denaturation of the enzyme and activity ceases.

3. Substrate concentration--- more reactions per second occur when the substrate is in very high concentration. Under these conditions, the active site is always occupied by a substrate molecule and the enzyme is said to be in saturation. Normal conditions in cells usually provide lower concentrations of substrate, so there may not be enough substrate molecules to give a maximum rate of reaction.

4. Inhibitors--- enzyme inhibitors slow or prevent the action of enzymes. Cells must be able to control the activity of enzymes.

a. Competitive inhibitors- these fill the active site so substrate molecules cannot reach it. The shape of a competitive inhibitor is close enough to the substrate so that it fits the active site, but it does not undergo any reaction while there. Competitive inhibitors can be:

1) Reversible- these inhibitors alternately occupy the active site and leave it. The enzyme can still function, but the rate is slowed.

2) Irreversible- inhibitor binds permanently to the active site, which ends the function of that enzyme molecule.

b. Non-competitive inhibitors---do not occupy the active site. The inhibitor binds to another area of the enzyme molecule, but this changes the shape of the active site so it can no longer function. This is called allosteric inhibition. It can be reversible or irreversible.

5. Enzymes that require metal ions as cofactors can be inactivated by chemicals that make the metal ions unavailable. (Cyanide poisoning)

FEEDBACK INHIBITION

This is a control mechanism that allows a cell to adjust the activity of enzymes to keep the amount of their product at an ideal level. Most substances needed in cells are produced in a series of reactions called a pathway. Each reaction in the pathway requires a separate enzyme. Example: a cell needs substance E. It cannot make this substance directly, in one easy step. However, by the following series of reactions, it can get E if it has substance A available as a starting point.

Enzyme # 1 Enzyme # 2 Enzyme # 3 Enzyme # 4

A B C D E

When so much E is made that it begins to accumulate, there is no need to keep making more, so the pathway needs to be temporarily shut down.

As this extra E accumulates in the cytosol, it comes in contact with Enzyme #1. It attaches to the enzyme molecule and causes allosteric inhibition. This shuts down the entire pathway.

As the extra E is used up, the E attached to Enzyme # 1 is released and Enzyme #1 becomes active, which starts up the pathway when E is needed.

RIBOZYMES

A fairly recent discovery is that there is a unique type of RNA, called a ribozyme, that can act as an enzyme. Ribozymes act only on RNA, removing sections and splicing the pieces together.

ENERGY PRODUCTION

Chemical bonds hold molecules together. Energy is required to build a bond. That energy is stored in the bond as long as it lasts, and the energy is released when the bond is broken.

The many small chemical bonds in nutrients are broken and their energy is used to build ATP. The unstable high-energy bond that attaches the third phosphate group of ATP is easily broken, releasing the energy to be used for the cell’s energy-requiring processes.

OXIDATION-REDUCTION REACTIONS

Oxidation—the removal of an electron from an atom or molecule

Reduction—the addition of an electron to an atom or molecule

Molecule A gives an electron to Molecule B

A is oxidized

B is reduced

Whenever one substance is oxidized, another substance must be reduced—the electron must have somewhere to go. This pairing is called oxidation-reduction or redox reactions. Biological oxidations often involve the removal of a hydrogen atom instead of just an electron alone. This can also be called dehydrogenation.

Cells use biological oxidation-reduction reactions to extract energy from nutrient molecules. Organic compounds with large numbers of hydrogen atoms make good energy sources for cells. The energy in glucose, for example, is released in a series of oxidation-reduction reactions and trapped in ATP.

GENERATION OF ATP

As ATP (3 phosphates) is generated, a phosphate group is added to ADP (2 phosphates). This third phosphate is attached by a high-energy bond—one with lots of energy that can be quickly released as this unstable bond is broken. Phosphorylation is the process of adding a phosphate group. Organisms do this in 3 ways:

1. Substrate-level phosphorylation—a high-energy phosphate is directly transferred from a phosphorylated compound (the substrate) to ADP.

C - C - C ~ P + ADP à C - C - C + ATP

2. Oxidative phosphorylation—electrons are transferred from organic compounds to electron carriers (usually NAD+ or FAD), passed through a series of electron carriers, and then finally to oxygen or some other inorganic molecule.

This process always involves a membrane—the plasma membrane of prokaryocytes and the inner mitochondrial membrane of eukaryocytes.

The series of electron carriers is called an electron transport chain. The transfer of electrons from one electron carrier to another releases energy and some of that energy is used to generate ATP. The process is called chemiosmosis.

3. Photophosphorylation—this occurs in cells that carry out photosynthesis. These cells contain light-trapping pigments (chlorophyll is the most common one). Light energy is converted to chemical energy (ATP) , which is then used to synthesize organic compounds. An electron transport chain is also involved in this process.

METABOLIC PATHWAYS OF ENERGY PRODUCTION

Organisms must release the energy stored in organic compounds in a series of step by step oxidation-reduction reactions. If the energy were all released at once, so much heat would be released that it would damage or destroy the cell. The series of reactions is called a metabolic pathway and each reaction in the pathway is catalyzed by a different enzyme.

CARBOHYDRATE CATABOLISM

Carbohydrates are the primary source of energy for living organisms, although lipids and proteins can also be used when carbohydrates are not available. Glucose is the fuel of choice, so carbohydrate metabolism is usually studied as glucose metabolism.

Energy is produced from glucose in 2 main ways:

1. Cellular respiration

2. Fermentation

Both processes begin with a series of ten reactions known as glycolysis or the Embden-Meyerhof pathway. Each of these reactions requires its own specific enzyme, and none of the reactions use oxygen (anaerobic).

In glycolysis, one 6-carbon molecule of glucose is converted to 2 3-carbon molecules of pyruvic acid. As the reactions proceed, there are 2 that require the input of ATP. A total of 4 molecules of ATP are produced by substrate-level phosphorylation, but since 2 ATPs are used, the net gain is 2 ATPs. This is only a fraction of the total energy contained in the glucose molecule.

Also, in this process, 2 molecules of the coenzyme NAD+ are reduced to NADH. We will see the importance of this shortly.

SEE FIGURE 5.12 P. 128 FOR THE STEP-BY-STEP REACTIONS OF

GLYCOLYSIS

Some bacteria are able to use other pathways in addition to glycolysis for the initial breakdown of glucose. These are:

a. Pentose phosphate pathway—can break down 5-carbon sugars as well as glucose. Products produced in this pathway are used as building blocks in biosynthesis.

b. Entner-Duodoroff pathway—mainly found in gram-negative bacteria. Bacteria with this pathway can use it instead of glycolysis.

As glycolysis ends, the 2 3-carbon molecules of pyruvic acid remain. More energy can be extracted from this pyruvic acid. Also, the reduced molecules of NADH cannot function again as coenzymes until they rid themselves of the hydrogens they have and return to the NAD+ form, so all cells must also have a way to accomplish this.

One possibility is that the pyruvic acid can be further broken down in the process of cellular respiration. This process always involves an electron transport chain and produces large amounts of ATP. It can be:

a. Aerobic—as the electrons are passed along the chain, the terminal electron acceptor is oxygen. Large amounts of oxygen are required.

b. Anaerobic—the terminal electron acceptor is some other inorganic molecule (not oxygen)

AEROBIC CELLULAR RESPIRATION

In aerobic cells, the next major step will be a series of reactions known as the Kreb’s Cycle (tricarboxylic acid or citric acid cycle).

Before this begins, a preparatory step will change the pyruvic acid left over from glycolysis to acetyl coenzyme A. One molecule of carbon dioxide is released and the remaining part of the pyruvic acid molecule, an acetyl group, combines with coenzyme A. Also, another NAD+ is reduced to NADH in this step. Since 2 pyruvic acids were produced from one glucose, 2 acetyl CoAs will be produced and 2 NAD+s will be reduced to 2 NADHs in this step for each glucose molecule.

As each acetyl CoA enters the Kreb’s Cycle, the molecule separates. Coenzyme A detaches and the acetyl group combines with oxaloacetic acid to form citric acid.

SEE FIG. 5.13 p. 130 FOR THE REACTIONS THAT MAKE UP

THE KREB’S CYCLE

As the Kreb’s cycle proceeds, the following are produced for every 2 acetyl CoAs:

4 CO₂

6 NADH

2 FADH₂

2 ATPs (substrate-level phosphorylation)

The reduced coenzyme molecules are very important, because they will be used as electron donors in the electron transport chain to produce large amounts of ATP. At the same time, by donating the hydrogen they had gained, they will be returned to the functional coenzyme form, NAD+, and be available to catalyze another reaction.

ELECTRON TRANSPORT CHAIN

This is a series of carrier molecules that will pass electrons along, releasing energy step by step, small amounts per step. This energy will drive the generation of ATP. An electron transport chain always involves a membrane:

Prokaryocytes—the plasma membrane

Eukaryocytes—the inner mitochondrial membrane

There are 3 classes of carrier molecules:

Flavoproteins--proteins that contain flavin, a coenzyme derived from riboflavin (Vit. B ₂)

Cytochromes--proteins that contain a heme group

Ubiquinones (Coenzyme Q)--small non-protein carriers

All electron transport chains achieve the same goal—releasing energy as electrons are transferred from higher-energy compounds to lower-energy compounds. At the end of the chain is an inorganic molecule that accepts the electron and does NOT pass it on (terminal electron acceptor). In aerobic cells (including human cells), the terminal electron acceptor is oxygen.

Various bacteria may have some variation in the exact carrier molecules present and the order in which they function, but the general idea is the same: reduced coenzyme molecules act as electron donors for the electron transport chain, and are returned to their functional form in the process.

Steps in the electron transport chain of eukaryocytes proceed as follows (this is the one we know the most about):

1. Electrons in the form of a hydrogen atom are passed from NADH to flavin mononucleotide (FMN), the first carrier in the chain: NADH is oxidized to NAD+ and FMN is reduced to FMNH₂

2. FMNH₂ pushes the protons (H+) to the other side of the membrane and passes 2 electrons to coenzyme Q

3. The electrons are next passed along the various cytochromes (5 different ones)

4. The last cytochrome (cyt a₃ ) passes the electrons to oxygen, which becomes negatively charged and attracts H+ to form H₂O.

5. FADH₂ also adds electrons to the chain, but at a lower level than NADH.

Some carriers transport hydrogen ions, but some transfer naked electrons only. When this happens, there is a proton released, which is pumped across the membrane by active transport. (This means the proton is pumped into the space between the outer and inner mitochondrial membrane in a eukaryocyte and out of the cell across the plasma membrane in a prokaryocyte.) This causes a buildup of protons on the outside of the membrane. This buildup of protons provides energy for the generation of ATP. The mechanism is known as chemiosmosis:

1. As electrons pass down the electron transport chain, some carriers in the chain pump protons across the membrane by active transport. These carriers are called proton pumps.

2. The membrane is impermeable to protons unless they are either pumped across by acti9ve transport or allowed to pass through channels specific to protons, so this establishes both a proton gradient and a charge gradient. This potential energy is called the proton motive force.

3. Protons can only cross back through the membrane through special protein channels that contain the enzyme adenosine triphosphatase (ATP synthase). This flow releases energy, which is used by the cell to synthesize ATP from ADP and P_i (inorganic phosphate).

In prokaryocytes carrying out aerobic cellular respiration, one glucose à 38 ATPs

To figure this, we need to know that:

Each NADH à 3 ATPs

Each FADH à 2 ATPs (because their electrons enter the ETC further along)

GLYCOLYSIS

4 ATP produced

- 2 ATP used à 2 ATP

2 ATP net gain

2 NADH à 6 ATP

PREPARATORY STEP

2 NADH à 6 ATP

KREB’S CYCLE

2 GTP (equivalent to) à 2 ATP

6 NADH à 18 ATP

2 FADH à 4 ATP

38 ATP

In eukaryocytes, the total is 36 ATPs, because 2 ATPs are lost when electrons cross into the mitochondrial membrane. (Remember, prokaryocytes do not have this membrane and use the plasma membrane instead.)

Overall:

C₆H₁₂O₆ + 6 O₂ + 38 ADP + 38 P à 6 CO₂ + 6 H₂O + 38 ATP

THIS IS ALSO SHOWN IN TABLE 5.3 P. 134 AND FIGURE 5.17 PAGE 135

ANAEROBIC CELLULAR RESPIRATION

A few anaerobic bacteria have electron transport chains that use an inorganic molecule other than oxygen as the terminal electron acceptor. This may be nitrate ions (NO₃), sulfate ions (SO₄^2-), or carbonate ions (CO₃^2-). The amountof ATP produced in anaerobic cellular respiration varies, but is always somewhat less than aerobic cellular respiration.

FERMENTATION

This is the process that follows glycolysis if the cell lacks an electron transport chain or if there is not enough of the terminal electron acceptor available to operate the chain. Most anaerobic bacteria must proceed this way after glycolysis. Facts about fermentation:

1. Releases a little more energy from sugars or other organic molecules

2. Does not require oxygen

3. Does not involve the Kreb’s cycle or an electron transport chain

4. Uses an organic molecule as the terminal electron acceptor

5. Produces only small amounts of ATP

Electrons are transferred from reduced coenzyme molecules to pyruvic acid or a substrate derived from pyruvic acid. In the process, the reduced coenzyme molecules are returned to their functional form for reuse.

Different microbes have the ability to ferment different substances. Pathways and end-products also vary. Tests of ability to ferment certain substrates and chemical analyses of end-products are sometimes used to identify microbes.

Two of the most important types of fermentation (named for their end products):

1. Lactic acid fermentation

1 molecule of glucose

¯ oxidized to

2 pyruvic acid molecules

¯ reduced by NADH

2 lactic acid molecules

This process can cause food spoilage, but it also produces yogurt, sauerkraut and pickles. The microbes get small additional amounts of ATP, but nowhere near the amount cellular respiration would produce.

2. Alcoholic fermentation

1 molecule of glucose

¯ oxdized to

2 molecules of pyruvic acid

¯

2 acetaldehyde molecules + 2 carbon dioxide molecules

¯ reduced by 2 NADH

2 ethanol molecules

Yeasts of the genus Saccharomyces that carry on alcoholic fermentation are used to make alcoholic beverages and to make bread rise.

Commercial products produced by fermentation include:

Vinegar

Cheese

Acetone

Glycerol

Methane

Vit C

LIPID CATABOLISM

Lipids are not a major energy source for most bacteria. However, some bacteria can produce enzymes called lipases that break down fats to glycerol plus fatty acids. These are metabolized separately, but both can eventually be put into the Kreb’s cycle.

Some bacteria can use the same enzymes to break down petroleum products. Beginning with natural ability and increasing it by genetic engineering, these are currently being used to help clean up oil spills.

PROTEIN CATABOLISM

Intact proteins cannot cross bacterial plasma membrane, so bacteria must produce extracellular enzymes called proteases and peptidases that break down the proteins into amino acids, which can enter the cell. Many of the amino acids are used in building bacterial proteins, but some may also be broken down for energy. If this is the way amino acids are used, they are broken down to some form that can enter the Kreb’s cycle. These reactions include:

1. Deamination—the amino group is removed, converted to an ammonium ion, and excreted. The remaining organic acid (the part of the amino acid molecule that is left after the amino group is removed) can enter the Kreb’s cycle.

2. Decarboxylation—the ---COOH group is removed

3. Dehydrogenation—a hydrogen is removed

Tests for the presence of enzymes that allow various amino acids to be broken down are used in identifying bacteria in the lab.

WHY does everything used to produce energy wind up in the Kreb’s cycle???

BIOCHEMICAL TESTS

Biochemical tests are frequently used in the lab to identify bacteria and other microbes. Morphology, gram-staining reaction, other special stains, etc. cannot come near identifying all existing types of bacteria, so biochemical tests are often required. These tests are really tests for the ability of the bacteria to make certain enzymes.

FERMENTATION TESTS

Liquid media containing a pH indicator and only one carbohydrate are used. A very small test tube is dropped upside down into the larger tube containing the liquid medium. Most fermentations result in the production of acid. The medium is adjusted to begin at a neutral pH. If the pH drops due to acid as an end-product of fermentation, this can easily be observed by the change in color due to the pH indicator. Although not as common as acid production, gas is sometimes produced, either alone or in addition to the acid. This is observed by examining the small test tube. If gas has been produced, some of it will accumulate as a gas bubble in the small tube.

Tests are also done for the ability to break down various amino acids.

PHOTOSYNTHESIS

Some organisms—plants and some microbes—have the ability to use light energy to synthesize complex organic substances (sugars) from simple inorganic substances. This is photosynthesis. Carbon dioxide gas is the source of the carbon, so this process is also called carbon fixation.

6 CO₂ +12 H₂O + LIGHT ENERGY à C₆H₁₂O₆ + 6 O₂ + 6 H₂O

Electrons are taken from water and incorporated into sugar. Energy from the sun is used.

Two stages:

1. Light reactions—light energy is used to convert ADP and phosphate groups to ATP (photophosphorylation). NADP is reduced to NADPH.

2. Dark (light-independent) reactions—electrons from NADPH plus energy from ATP are used to reduce carbon dioxide to sugar (Calvin-Benson cycle).