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The growth and reproduction of bacteria entails many chemical reactions that are collectively called metabolism. This lecture will attempt to briefly describe the principal features of metabolism together with some energy reactions that are involved in biological processes.
The capacity to utilize and transform energy in one of the most fundamental properties of living organisms. In this lecture we are also concerned with the basic forms of energy recruitment, namely,
(1) Photosynthesis
(2) Fermentation & Respiration.
Light is a primary source for photosynthesis and hence the process photophosphorylation. Energy obtained from light can be used by non-photosynthetic organisms as well.
(3) Chemical energy
(fermentation and respiration) is the energy that can be released from inorganic and organic compounds by a chemical reaction and is the primary energy for the non-photosynthetic organism as well. The use of a chemical substance as an energy source always involves what is called an oxidation-reduction reaction (OR). That is, the energy source becomes oxidized, while another substance is reduced. Although some (OR) reactions require oxygen, many do not, instead of oxygen transfer, the real basis of an (OR) system is electron transfer.
A key component of an (OR) is nicotinamide adenine dinucleotide (NAD). Since it has the ability to be alternately oxidized and reduced it carries electrons from an organic source to the electron acceptor. The coupling of NAD with an OR reaction results in the synthesis of high-energy phosphate bonds in adenosine triphosphate (ATP). As we shall see in the following discussion the energy of ATP is used by the cell in various biosynthetic reactions of the cell that in turn leads to new cell material and growth. Thus, ATP can be viewed as a sort of energy currency for the cell. Nevertheless, ATP is a short-lived energy reserve, therefore for long-term energy storage, organic polymeric compounds such as starch and glycogen are formed.
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The maintenance of viability, growth, reproduction and other activities of the bacterial cell are accomplished through an intricate system of reactions by enzymes. Enzymes are organic catalysts which have the capacity to "speed up" certain chemical reactions without themselves being changed by the reaction. They accelerate the velocity of the reaction without necessarily initiating it. For example, under normal atmospheric conditions, hydrogen and oxygen do not combine to any appreciable extent. If, however, the two gases are allowed to contact colloidal platinum, they react instantaneously to produce water. Platinum greatly increases the speed of the reaction that takes place without being used up in the reaction. However, unlike platinum which is inorganic, enzymes are organic compounds produced by the living cell.
Two types of enzymes are recognized on the basis of their site of action: intracellular enzymes or endoenzymes and extracellular enzymes or exoenzymes. The main function of the extracellular enzymes is to perform whatever changes are necessary on the nutrients in the medium to allow food to enter the cell. The intracellular enzymes synthesize cellular material and perform catabolic reactions providing energy requirement by the cell. Enzymes may be regarded as vital part of the cell, since impairment of their activity is reflected by change in the cell, even to the point of death.
The term metabolism denotes the organized chemical activity performed by two types of reactions:
(1) Catabolism or dissimilation--the breaking down or degradation of the substrate; and(2) Anabolism or assimilation--the synthesis or building up of material by the cell.
Hence, by means of metabolism, some molecules of substrate are reduced in size and complexity, and new substances are synthesized to repair old cells or produce new ones.
Energy Relationship: If it is to remain alive and grow, the bacterial cell, like all other cells, must be capable of doing work. One of the important results of the chemical activity of the cell is that of providing energy. In accumulating high levels of substances within the cell environment, bacteria require many energy functions.
Chemical reactions are designated as exergonic (energy yielding) or endergonic (energy requiring) on the basis of energetics. In essence, some reactions will not occur unless some special provision is made to increase energy content of the reactants, as is shown in the following generalized equation.
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Substrate Product
The above reaction proceeds in the direction of the arrow since the sum of the chemical energy of A + B is greater than that of C + D.
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Substrate Product
The sum of the chemical energy of E + F is less than that of the products, G + H. Hence, to make the reaction go in the direction of the arrow, more energy is required at the substrate level.
The required energy made available is an "activated" (high energy) form of one of the reactants to "drive" the reaction in the desired direction. Assuming F in the above equation has been activated to a high energy level, the sum of the chemical reaction of E + F (high energy) is greater than that of G + H; therefore, the reaction will proceed in the direction indicated. The chemical reaction associated with catabolic reactions varies extensively as to the amount of energy released. Likewise, chemical reactions involved in anabolism are endergonic and thus exhibit energy requirements different from catabolism.
The metabolism of most cells is bound up with the primary goal of obtaining enough energy for cellular metabolism and growth. Excess energy is stored, up to a point, in various high energy compounds, among which are found fat and oil molecules as well as starch and other polysaccharides. A part of the general metabolic protein pool may also be used in times of need as an energy source.Many and varied are the molecules which some bacteria can turn to as a source of energy.
Bacteria can be divided into many groups. The primary grouping lies in the differentiation between autotrophic and heterotrophic metabolism.
(1) Autotrophic (self-feeding) metabolism lies in two major categories: that of photosynthesis which derives energy from the respiration of inorganic electron donors.(2) Some species may use simple organic compounds when available. Therefore, the definition of an autotroph is best defined as an organism utilizing CO2 as a prime source of energy.
Microbial photosynthesis, mostly by algae, accounts for about half of the total terrestrial photosynthesis. The process is rather simple. A quantum of light when absorbed by a chlorophyll molecule displaces an electron. This causes a positive (oxidizing) region and a negative (reducing) region in the chlorophyll molecule. The positive charge is promptly neutralized by taking up an electron from a closely-linked cytochrome molecule (Fe+, Fe+++) of high redox potential while the negative charge is transferred from the chlorophyll molecule to a cofactor of very low redox potential, ferredoxin. These two very widely differing factors, cytochrome and ferredoxin, constitute the ends of the electron chain through which the potential difference is used to create metabolically useful energy (ATP) and reducing power (NADH). This simple transport system can function in two ways to create different proportions of ATP and NADH as needed. In cyclic photophosphorylation, the electron is transported from the reduced factor to the oxidized cytochromes, and ATP is similarly generated. In the noncyclic photophosphorylation process, which produces useful power as well as ATP, the electron at the reducing end of the chain is trapped by reducing NAD of the parallel enzyme NADP to complete the electron transport with an electron supplied from another source. In algae and other plants, it is derived from the oxidation of water.
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Bacteria are apparently unable to generate the very high potential necessary for this oxidation, so a more readily oxidized donor of electrons must be available. Hydrogen sulfide (H2S) and CO2 are common donors. Thus bacterial photosynthesis never releases O2, but some of the purple sulfur bacteria release an equivalent amount of H2SO4 or S.
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^ ^ ________________ H2S -----------> 2H+ + S
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is the response of the bacterium to a gradient of light intensity which causes differential flagellar activity resulting in motion towards the light. See Figure 1 (Handout).
Many bacteria can use nitrogen in place of oxygen as a final hydrogen acceptor. The conversion of molecular nitrogen into nitrogenous compounds is known as nitrogen fixation. Two groups of bacteria are involved in this process:
(1) Nonsymbiotic microorganisms--those living freely or independently in the soil; and(2) Symbiotic microorganisms--those living in roots or leguminous plants. The most important agents of nonsymbiotic nitrogen fixation are blue-green algae of the types Anagaena and Nostoc and aerobic bacteria of the genus Azotobacter. Many other bacteria (e.g., photosynthetic bacteria, Clostridium spp., Bacillus polymya) can also fix nitrogen.
Symbiotic nitrogen fixation is accomplished by bacteria of the genus Rhizobium in association with legumes (plants that bear seeds in pods, i.e., soybean, clover, peas). Bacteria must establish themselves in the roots of plants before they can fix nitrogen. A summary of the various transformations involving nitrogen and nitrogen compounds are shown in Figure 2 (Handout).
Heterotrophic metabolism means feeding on others, literally. Heterotrophs live on preformed organic compounds. We will now discuss several forms of bacteria as to their requirements for molecular oxygen: obligate respiratory organisms (aerobes), obligate fermentative organisms (anaerobes), and facultative organisms. Facultatives grow either with or without oxygen. When O2 tensions change, these bacteria have only fermentative enzymes; however, they are able to ferment substrates in the presence of oxygen, and they continue to do so.
Respiratory bacteria are more common than fermentative bacteria. Why? Respiration is simply more efficient then fermentation. It yields about 10 times more free energy from glucose, and it also converts more of this free energy to ATP. Cellular respiration converts 45% of the total free energy released to ATP, while fermentation converts only about 25%. The rest of this free energy appears as heat, which in large scale fermentations must be dissipated if the reaction is not to be sterilized in its own heat.
Obligate Aerobes, such as Mycobacterium and some spore forming bacillus require oxygen and lack the capacity for substrate fermentation with regard to the effect of oxygen on their growth and metabolism.
Obligate Anaerobes, such as Clostridium, grow only in the absence of oxygen. (Microaerophilic organisms tolerate oxygen at a low tension, but not like air.)
Facultatives can be grown with our without O2, such as yeast or E. coli.
The opposite of an obligate anaerobe is the obligate aerobe. These bacteria will not grow at a markedly reduced oxygen tension. They have only one mode of energy metabolism, respiration.
Respiration can be defined as the energy producing oxidation of a substrate at the expense of molecular oxygen.
To serve as an energy source, organic compound must be willing to give up electrons and become oxidized. Since each (OR ) must be accompanied by a reduction reaction there must be an electron acceptor to take up the electron from the energy source. The most widely occurring electron acceptor is O2, but oxygen can not serve as an electron acceptor unless it is activated by a cytochrome system present in an electron transport particle. When O2 accepts an electron it becomes reduced to H2O. The utilization of O2 as an electron acceptor is known as respiration. Other inorganic compounds such as nitrate, ferric iron, sulfate and CO2 can replace O2 as an acceptor. Utilization of these electron acceptors in place of O2 is called anaerobic respiration.
The electron transport chain is respiration, for it is the means by which electrons flow from NADH to oxygen. The oxidative phosphorylation which accompanies this is shown in Figure 3 (Handout).
Much of the energy released by the transfer of electron to NHDH2 to O2 is conserved through the production of ATP. Within the electron transport system by a process called oxidative phosphorylation. In oxidative phosphorylation, ATP synthesis is coupled with electron transport and oxygen uptake.
The rate of oxidative phosphorylation is studied experimentally by measuring the rate of oxygen uptake and the rate of phosphate conversion to ATP. The rate of phosphate uptake often referred to as the P/O is then calculated. When NADH2 is the electron donor, the ration is about 3, that's 3 molecules of ATP are synthesized for each atom of oxygen taken up and each mole of NADH2 oxidized. In electron donors other than NHDH2 the P/O ratio may differ. For example, the succinate oxidation, which involved the direct donation of electron by succinate to flovoprotein without thee mediation of NAD, the P/O ratio is 2. The most important aspect of oxidative phosphorylation is that is provides the organism with a means of deriving energy from oxidation of NADH2. However, oxidative phosphorylation is contingent on the presence in the environment of oxygen gas, or other suitable electron acception.
The electron transport systems have two basic functions:
(1) To accept electron from the electron donor and transfer them to electron acceptor (O2), and(2) to conserve some of the energy that's released during electron transfer by the synthesis of ATP. Key components of electron-transport systems are the flovopriteins and cytochromes which act as electron carrier.
Flavoproteins are proteins containing a derivation of riboflavin. The flavin portion which is bound to a protein is the prosthetic which is alternatively reduced when is accepts electrons and oxidized when the electrons are passed on.
The cytochromes are iron-containing porphyrin rings attached to proteins. They undergo oxidation and reduction through loss or gain of an electron by the iron at the center of the cytochrome.
Another major biochemical pathway for handling "sugars" is fermentation. Phylogenetically fermentation is older than respiration, although much less efficient. Fermentation is a process which leads to many organic products such as wine, beer and cider. The list is long and was well known to ancient man, although modern science has had a hand in increasing the items thereon.
When organic compounds are utilized as energy sources in the absence of O2 or an inorganic electron acceptor the process is called fermentation. In this process, organic compounds serve as both electron donors and electron acceptors. For instance, in the fermentation of glucose by yeast, some atoms of the glucose molecule are oxidized to CO2, whereas others are reduced to alcohol. Although NAD is involved in fermentation, a cytochrome system is not. In respiration, all of the potential energy of an organic compound can be released, in fermentation it can not. Therefore, energy and ATP yields in fermentation are much lower than in respiration.
Fermentation is defined as metabolism in which energy is derived from the use of organic compounds as both the electron donors and electron acceptors. There are a variety of fermentation processes, leading to many different products; however, they are quite similar to pyruvate. These form, then, the glycolytic pathway, shown in Figure 4 (Handout).
A variety of fermentations, yielding quite different products, are based on the glycolytic pathway, which is present, up to pyruvate, in the accompanying diagram (Figure 5 of handout). Adenosine triphosphate generation in this pathway depends on the oxidation of triose phosphate to an acid, at the expense of the reduction of NAD+. Since the total NAD in the cell is very limited, fermentation would cease very rapidly if the reduced NAD were not reoxidized in a further reaction of pyruvate. Microbes have evolved a variety of pathways for this purpose, some of which yield additional ATP. The principal pathways of fermentation shown in Figure 5 (Handout) are:
1. Lactic acid (Streptococcus lactobacillus)2. Alcoholic (many yeast, few bacteria).
3. Mixed Acid (Enterobacteraceae, including Aerobacter)
4. Butanediol (Aerobacter)
5. Butyric (Clostridium)
6. Propionic (propionic acid bacteria)
Our first concern will be energy metabolism. We will begin with the study of the metabolism of the various carbohydrates and starches and alcohols which are referred to as "sugars". There are 30 sugars commonly used in bacteriology. They are:
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These substrates yield energy to the cell in spite of their varying structure. This is due to the theory unity of biochemistry. This theory states that there are certain common metabolic pathways followed by all organisms. The chief factor lies in knowing the difference between essential nutrients for a cell and essential metabolites. The list of essential nutrients varies widely from the photosynthetic algae which need sunlight, water and some salts in solution to the nutrition of a human being in a space capsule. The essential metabolite, however, differ very little on a qualitative basis.
Heterotrophic bacteria have been studied more extensively than the autotrophs. This is apparent because, in a sense, heterotrophs are of the most immediate concern to human kind. In this group of bacteria we find the species that cause diseases of human being, other animals, and plants as well as the greater part of the microbial population in man's immediate environment. This does not mean that autotrophs are less important. On the contrary, they are of utmost importance in less conspicuous but indispensable processes in nature such as the cycling of elements through biological systems as shown in the activities of microorganisms in soil and water.
Although heterotrophic bacteria constitute one major nutritional group, they vary considerably in the specific nutrient required for growth, see Table EMJ(4)-1. All heterotrophs require an organic form of carbon; however, they differ as to the kind of organic carbon compounds they can utilize. All of the heterotrophs utilize CO2; however, CO2 alone will not satisfy their carbon requirement as it does for the autotrophs. In addition, with respect to nitrogen requirements, even greater diversity is exhibited. Some heterotrophs are satisfied with atmospheric nitrogen compounds, while still others require one or more organic compounds. Some heterotrophs will grow in a medium containing vitamins; others require one or more vitamins or vitamin-like substances.
Depending on the species, heterotrophic bacteria may have relatively simple or complex nutritional requirements. This is shown more specifically in Table EMJ(4)-1, where media for the growth of Escherichia coli and the Lactobacilli are compared. Note the striking difference in the number and complexity of the chemical nutrients required for growth of the two species even though both genera are heterotrophic.
Amino acids are also fermented. Although fermentation products of proteolysis lack the economic significance of the fermentations of pyruvic acid, they are of medical importance. They are prominent in putrefactive processes, including gangrene associated with anaerobic wound infections. These fermentations occur where there is considerable proteolysis and certain amino acids or their delamination products serve as electron donors and acceptors. For example,
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As can be seen, one of the products of this reaction is NH3. The decarboxylation increases the volatility of the products which also tend to be strongly odorous nitrogenous compounds. One such amino acid fermentation often employed in the laboratory is the formation of indol, a breakdown product responsible for minor flavoring of beer, wine and cheese. Unfortunately, one of the longer chain products of this reaction, called fusel oil, may be a major constituent of a hangover.
The fermentative reactions take place without the presence of oxygen. Indeed many bacteria which have a primarily fermentative metabolism cannot survive in the presence of this element. The bacterial metabolic requirement is termed obligate anaerobiasis. Although it is uncertain why oxygen prevents the growth of obligate anaerobes, the most logical choice is its effect in maintaining some enzymes in an oxidized state which prevents their carrying out their reducing function.
Some obligate anaerobes are not inhibited by oxygen, they are killed. Here again the mechanism is unknown; however, it is well known that these species lack the enzyme catalase which enhances the reaction 2H2O2 --> 2H2O + O2. Although there are a few bacteria which instead of eliminating H2O2 utilize it by means of certain peroxidases, they are rare, and the most probable result of a lack of catalase in an oxygen-rich environment is biochemical suicide. Another, and perhaps more common, subgroup of these anaerobic bacteria is the microaerophiles. These bacteria tolerate or require very small quantities of O2, but are completely inhibited by concentrations approaching that of the atmosphere.
The major energy-yielding pathways when first discovered were considered to be entirely catabolic or degradative. Further examination shows that these pathways of glycolysis, pyruvate oxidation and tricarboxylate (TCA) cycle function just as directly and indispensably in biosynthesis as in energy production. Because their original designation as catabolic pathways is not true, it has been proposed to substitute the term amphibolic for those pathways which can perform either function, while the term anabolic is retained for those short branches committed to energy metabolism or restricted to the short sequences that convert amphibolic intermediates to fermentation end products. Biosynthesis is a branch of quite a variety of the intermediates in the so-called catabolic pathways.
4.15.1 Biosynthesis from 2-C Compounds: The Glyoxylate Cycle
The pathways (see handout) that have been reviewed can account for the ability of organisms to grow on many compounds: sugars (or substances convertible to them), 3-C monocarboxylic acids, 4 to 6-C di- or tricarboxylic acids, and even CO2 in autotrophs. These pathways, however, do not account for the ability of many bacteria to grow on acetate, and hence on fat, as the sole C source. While pyruvate is readily decarboxylated to acetate, the reverse reaction, like the reverse decarboxylation of alpha-ketoglutarate does not occur (except in some photosynthetic organisms). Moreover, acetate cannot condense with itself "back-to-back" to yield succinate. This inability of acetate to replace the biosynthetic drain on the TCA cycle is largely responsible for the acidosis of a diabetic mammal forced to consume fat (via acetyl CoA) without carbohydrate. So the question remains, how can bacteria thrive on such a diet? See Figure 7 (Handout).
The answer is a bypass or epicycle on the TCA (Kreb's) Cycle, involving two additional enzymes. These enzymes catalyze reactions (1) and (2) on next page:
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The succinate and malate formed in (1) and (2) can be used to regenerate isocitrate through familiar reactions of the TCA cycle, summed as reaction (3), yielding a glyoxylate cycle. This cycle shares part of the enzymes of the classic TCA cycle but bypasses its two decarboxylative reactions above. Acetate is thus used for net synthesis of 4-C compounds rather than burned to CO2.
The two enzymes of the glyoxylate bypass have been found in varieties of organisms grown on acetate as a sole C source, but their formation is repressed by the simultaneous supply of a more rapidly used substrate, such as glucose or succinate. This response resembles the diauxic response to different sugars.
The tricarboxylic acid cycle (TCA cycle), discovered by Sir Hans Kreb and often called the citric acid cycle, is utilized by most aerobic organisms to oxidize pyruvic acid completely to CO2. The NADH2 formed in the TCA cycle is reoxidized by way of the electron transport system with concomitant production of ATP by oxidative phosphorylation.
Pyruvic acid is first decarboxylated, leading to production of one molecule of NADH2 and an acetyl radical coupled to Coenzyme A (CoA). Acetyl-Coenzyme A (abbreviated acetyl-CoA) is an activated form of acetate, the acetyl-CoA bond being the highest energy bond. In addition to being a key intermediate in the TCA cycle, acetyl-CoA also plays many important biosynthetic roles. The acetyl group of acetyl-CoA combines with the 4-C compound oxaloacetic acid, leading to the formation of citric acid, a 6-C organic acid, the energy of the high-energy acetyl-CoA bond being used to drive the synthesis. Dehydration, decarboxylation, and oxidation reactions follow, and two CO2 molecules are released. Ultimately oxaloacetic acid is regenerated, and can serve again as an acetyl acceptor, thus completing the cycle.
For each pyruvate molecule entering the cycle, three CO2 molecules are released, one during the formation of acetyle-CoA, one by the decarboxylation of isocitrate, and one by the decarboxylation of alpha-ketoglutarate, see Figure 8 (Handout).
The net result of the TCA cycle is the complete oxidation of pyruvic acid to CO2 with the production of four molecules of NADH2. Each of the NADH2 molecules can be oxidized back to NAD through the electron transport system, producing three ATP molecules per molecule of NADH2 oxidized. In addition, the oxidation of succinyl-CoA to fumaric acid involves substrate-level phosphorylation, producing GTP which is later converted to ATP, and this oxidation also involves donation of electrons to the flavin of an electron transport particle without the mediation of NAD, producing two or more molecules of ATP for a total in the two reactions of three ATPs. Finally, two molecules of ATP are produced by substrate level phosphorylation during the conversion of glucose to pyruvic acid, so that aerobes can form 38 *atp molecules from glucose breakdown, in contrast to the two molecules of ATP produced anaerobically.
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The ability to produce light is a property of a number of living organisms, including some bacteria, fungi, and algae (dinoflagellates). Among higher organisms, bioluminescence occurs in fireflies, and in some jellyfish and crustaceans. The detailed mechanism of light production is not the same in all these organisms, but in all cases it involves energy transfer and production of an excited state in a molecule that, upon returning to the ground state, emits light. Luminescence by a bacterial culture is shown on next page.
4.16.1 Pathway of Electron Flow in Bacterial Luminescence
NADH2
v
FMN > Luciferase
(Flavin mononucleotide)
v
Cytochromes
v
O2, aldehyde
Activated luciferase
v
O2
v
Luciferase + Light
The biochemistry of bacterial luminescence has been studied in cell-free extracts. Two specific components are the enzyme luciferase and a long-chain aldehyde (like dodecanal); flavin mononucleotide and O2 are also involved. The primary electron donor is NADH2.
The aldehyde is not required for reduction of the enzyme, but in the absence of the aldehyde the amount of light emitted when the activated enzyme returns to the ground state is low.
As shown, bioluminescence competes with normal electron transport for the electrons of NADH2. One consequence of this competition is that, if the activity of the cytochrome system is blocked by cyanide or some other inhibitor, the intensity of luminescence is increased.
STUDY QUESTIONS Define the following:
- autotrophic syntrophism oxidative
- phosphorylation
- heterotrophic respiration cytochrome oxidase
- photosynthesis glycolytic pathway P/O ratio
- cytochrome amino acid fermentation tricarboxylate cycle
- bioluminescence
- redox potential indol anaerobic
- phototaxis Tryptophane anaerobic
- respiration
- nitrogen cycle fusel oil facultative
- bacteria
- Rhizobium obligate anaerobes catabolic
- pathway: "Sugars pathway"
- fermentation catalase amphibolic
- pathway
- peroxidase anabolic
- Unity theory of Biochemistry chemautotrophy ATP
- essential nutrient microaerophile ADP
- essential metabolite obligate aerobe endergonic
- autotrophic mutant electron transport chain exergonic
- Diagram and label the following biochemical pathways:
- Cyclic photophosphorylation
- Noncyclic photophosphorylation
- Glycolytic pathway (as far as pyruvate)
- Lactic acid fermentation (from pyruvate on)
- Alcoholic fermentation (from pyruvate on)
- Mixed acid fermentation (from pyruvate on)
- Butanediol fermentation (from pyruvate on)
- Butyric fermentation (from pyruvate on)
- Propionic fermentation (from pyruvate on)
- Amino acid fermentation
- Catalase reaction
- Electron transport chain in relationship to glycolytic pathway
- Pyruvate oxidation
- Tricarbosylate cycle
- Bioluminescence equation
1. Diagram the electron transport chain in the relationship to the glycolytic pathway.
2. Diagram the tricarboxylate cycle and label points of energy yield as ATP.
3. Discuss the differences in energy economy between respiration and fermentation.
4. Define the following terms: enzyme, apoenzyme, holoenzyme, enzyme prosthetic group, anaerobic, aerobic.
5. Distinguish between adoptive, induced, and constituted enzyme.
6. Discuss some factors that influence enzyme activity.
7. Explain how one can demonstrate experimentally that a particular enzyme is intracellular or extracellular?
8. Write a word equation illustrating the reaction brought about by enzymes.
References: Microbiology-Davis, Dulbeco, Eisen, Ginsberg and Wood. 4th edition, 1990.
