303 Bacterial metabolism 2.docx

**Bacteria Metabolism** **Enzymes and metabolism** Metabolism is the sum of all biochemical processes taking place in bacteria and other living things. Metabolism is divided into 2 major categories; 1. Anabolism which is the synthesis of organic compounds 2. Catabolism, the breakdown of these...

**Bacteria Metabolism** **Enzymes and metabolism** Metabolism is the sum of all biochemical processes taking place in bacteria and other living things. Metabolism is divided into 2 major categories; 1. Anabolism which is the synthesis of organic compounds 2. Catabolism, the breakdown of these compounds. For anaboliosm and catabolism to take place, enzymes must be present. Enzymes are a group of organic molecules (usually proteins) that bring about chemical changes while themselves remain unchanged. They speed up chemical processes by achieving in seconds what would have otherwise taken hours, days or even longer to happen. Enzymes are reusable. Once a chemical reaction has occurred, the enzymes are released to take part in other reactions. The number of enzymes in bacteria are small compared to number of reactions that occurs within the bacterial cells. Enzymes are highly specific, an enzyme that takes part in one reaction usually will not participate in another type. The substance that the enzyme works on is called the substrate and the products produced in the reaction are called end-products. Since many reactions are reversible, enzymes may bring about synthesis (building -up) as well as digestions (breaking down). In order for synthesis or digestion to take place, certain important areas called active sites must be present and available on the enzyme molecule. Active groups often contain sulfhydryl groups (-SH), so any substance reacting with sulfhydryl groups will tie up the groups and prevent enzymatic action. Silver, a heavy metal acts in this fashion, it binds to the sulfhydryl groups thereby preventing the bacteria from performing even their most basic functions. Silver is useful as a disinfectant in such forms as silver nitrates. Another way of inactivating an enzyme is by blocking its active sites with a compound closely related to the normal substrate. Sulfonanide drugs operate this way. Many enzymes are named after the substance they break for example lactase breaks down lactose, sucrase breaks down sucrose, ribonuclease digests ribonucleic acid. Because enzymes are mainly proteins, they are very sensitive to physical or chemical agents. Heat denatures the tertiary structure of enzymes, alcohol precipitates enzymes proteins and therefor can be used as chemicals. Some enzymes are made up entirely of proteins example lysozyme and many other made up of proteins and non-protein such as an ion of magnesium, iron or zinc which are called cofactors. When the non-protein part is organic molecule, it is called a coenzyme. Examples of co enzymes include adenine dinucleotide (NAD) and flavin adenine dinucleotide, **Energy and ATP** In all living things, molecules move about constantly leading to chemical reactions. Certain chemical reactions yield energy but in many cases **inactivation energy** has to be supplied for other energy yielding reactions to take place**.** Enzymes play a key role in metabolism because they lower the amount of activation energy required for a reaction to take place. They assist in the breaking down of chemical bonds and making of new ones by separating and joining atoms in a timely manner. The reactions would probably take place without enzymes but they will much more slower and far less efficiently. In metabolism, enzymes require the chemical energy in a compound called **adenosine triphosphate (ATP).** A molecule of ATP can be compared to a portable battery as it moves to every part of the cell where an energy consuming reaction is taking place and provides energy. In bacteria, ATP supplies energy for binary fission, flagellar motion and spore formation. It fuels protein synthesis and carbohydrate breakdown. The energy in ATP is releases by breaking the high energy bond holding the last phosphate group to the molecule to produce adenosine diphosphate and a phosphate group. This reaction is catalysed by an enzyme called adenosine triphosphatase (ATPase). The energy formerly locked up is now set free to work. A single molecule of ATP releases 7300 calories of energy when its bonds are broken. A picture containing diagram, design Description automatically generated **Figure 1: Hydrolysis of ATP** Although ATP molecules are used everywhere in a bacterium cell to meet energy needs, they are not suitable for storing energy. The molecules are unstable, and any surplus takes up so much space in a cell. A mole of ATP weighs 507 grams. Therefore, cells synthesize or obtain small molecules such as glucose or lipids for energy storage. Later on the energy stored up as glucose or lipids are converted are release through catabolism and used to reform ATP from adenosine diphosphate (ADP) and phosphate. The new ATP is then used to drive the reactions of metabolism and other activities of the bacterium. In summary **ATP is an Immediate Energy Source** - **Bacteria cannot directly get its energy from sugars (glucose).** **Glucose** is not a **direct** energy source. Instead, the bacteria use the energy released from breaking down glucose, to drive phosphorylation of ADP. This makes **ATP**, which is an **immediate** energy source that cells can use quickly. - **Energy is stored in the bonds joining phosphate groups**. The energy in ATP molecules is stored within the **phosphoanhydride bonds **(high energy between the three phosphate groups. To release this energy, the bond must be broken. This happens through hydrolysis which we are yet to discuss. - **The phosphate bonds are broken by hydrolysis**. Hydrolysis of ATP forms ADP (adenosine diphosphate) and an inorganic phosphate group (Pi). This hydrolysis is catalysed by the enzyme **ATP hydrolase**. ATP hydrolase can further catalyse ADP into adenosine monophosphate (AMP) and a second inorganic phosphate group. **Catabolism** **Cellular Respiration in Bacteria** The catabolism of a glucose molecule does not take place in one chemical reaction nor do the ATP molecules (38 molecules of ATP) form all at once. The process involves one or more steps which forms a metabolic pathway. A metabolic pathway is a sequence of chemical reactions usually catalysed by enzymes in which one product of one reaction serves a s a substrate for the next reaction. Respiration is a series of biochemical reactions in which energy is released. Glucose catabolism is form of respiration. Respiration and catabolism are often used interchangeably. Respiration may occur in the presence of oxygen in which case it is called aerobic respiration. In other instances, it may take place in the absence of oxygen, in which case it is called anaerobic respiration or fermentation. Glucose catabolism can take place by both methos, aerobic respiration or fermentation. The numerous metabolic pathways for aerobic respiration can be represented as in summary follows C~6~H~12~O~6~ + 6O~2~ + 38ADP →6CO~2~ +6H~2~O + 38ATP **Cellular respiration** is therefore a process that uses oxygen, nitrate, or sulfate to break down nutrients to generate a cell\'s energy. In the process of breaking down nutrients such as glucose in aerobic respiration, carbon dioxide and water are generated. Carbon dioxide and water are waste products of aerobic cellular respiration. Cells, including bacteria, can be thought of as energy producing factories that take nutrients and convert them into energy called adenosine triphosphate (ATP). Some cells may be better than others at producing ATP because they use more efficient methods. The benefit of using cellular respiration for bacteria is the amount of energy or ATP generated. The reason cellular respiration generates so much ATP is because it maximizes the use of glucose by using by-products generated in other energy producing pathways. **Glycolysis** The chemical breakdown of glucose is called glycolysis. The term \'glycolysis,\' \'glyco-\' means sugar, and \'-lysis\' means to break. **Glycolysis is the breakdown or the catabolic process of converting one molecule of glucose into two molecules of pyruvate**. Glycolysis can be defined as a sequence of 10 chemical reactions that breaks down glucose into pyruvate, releasing energy that is then captured and stored in ATP. It is the central pathway for glucose catabolism and is found in both aerobic and anaerobic organisms. Glycolysis is an ancient metabolic pathway and is found in the great majority of organisms alive today. It is the first step in respiration, where glucose is oxidized to a simpler organic compound, releasing energy that is used to produce ATP from ADP4. Glycolysis is the first step in breaking down glucose to obtain energy through cellular respiration in bacteria. There are several metabolic pathways for glucose to be broken down, but the best way know is called Embden-Meyerhof pathway after Gustav Embden and Otto Meyerhoff, two German biochemists who described the pathway in detail in 1930. This set of reactions occurs in the cytoplasm of bacteria and involves the conversion of glucose to a 3-carbon organic compound called pyruvic acid or pyruvate. Between glucose and pyruvate are nine chemical reactions (but the whole pathway consists of 10 steps) and each reaction of step is catalysed by a specific enzyme. The product of glycolysis, **pyruvate**, can be broken down further to generate even more energy. Even though glycolysis is a larger process of aerobic respiration, glycolysis only occurs in the absence of oxygen. Glycolysis does not require oxygen and many anaerobic organisms use this pathway.  **Figure 2: Glycolysis by the Embden-Meyerhof Pathway** Glycolysis takes place in the cytosol of a cell, and it can be broken down into two main phases: the energy-requiring phase, and the energy-releasing phase. - **Energy-requiring phase.** In this phase, the starting molecule of glucose gets rearranged, and two phosphate groups are attached to it. The phosphate groups make the modified sugar---now called fructose-1,6-bisphosphate---unstable, allowing it to split in half and form two phosphate-bearing three-carbon sugars. The three-carbon sugars formed when the unstable sugar breaks down are different from each other. Only one---glyceraldehyde-3-phosphate---can enter the following step. However, the unfavourable sugar, DHAP can be easily converted into the favourable one, so both finish the pathway in the end. - **Energy-releasing phase**. In this phase, each three-carbon sugar is converted into another three-carbon molecule, pyruvate, through a series of reactions. In these reactions, two ATP and one NADH are made. Because this phase takes place twice, once for each of the two three-carbon sugars, it makes four ATP and two NADH overall. In Glycolysis, one 6-carbon glucose molecule is finally converted into two 3-carbon pyruvic acid molecules. For the conversion to occur, two molecules of ATP must be supplied. One molecule is used up at the beginning of glycolysis (1) and the second one is needed for reaction (3). Reaction (1) gives us glucose-6-phospahte and the reaction (3) gives fructose 1-6-diphosphate (di means 2 phosphate molecules). An important split reaction occurs in reaction (4). The fructose 1-6-diphosphate molecule breaks to yield two molecules, dihydroxyacetone phosphate (DHAP) and phosphoglyceraldehye (PGAL). PGAL is also called glyceraldehyde-3-phosphate. An enzyme converts DHAL molecule to another PGAL molecule. Each PGAL molecule passes through a series of conversions and finally forms pyruvic acid. These reactions occur throughs steps (6) to (10). A significant event takes place in reaction (7). As the enzyme conversion proceeds, enough energy is released to synthesize an ATP molecule from ADP and phosphate in a coupled reaction. This chemistry happens again in reaction (10). Each time a PGAL molecule is broken down to pyruvic acid through the sequence, two ATP molecules are formed. Since there are two PGAL molecules available, a total of four molecules of ATP are formed. Considering that two ATP molecules were invested at the beginning of the glycolytic pathway, the net gain of ATP in glycolysis is 2 molecules of ATP. In reaction (6), two high- energy electrons and 2 protons are released and shuttled to NAD converting it to NADH. In glycolysis, the cell gains two molecules of ATP for use in its metabolism even though no oxygen has been utilised. Two possible outcomes for pyruvate are the Krebs cycle or fermentation. Fermentation generates some energy for the cell, but it also generates toxic products like alcohol, acetic acid (or vinegar), and lactic acid that have to be cleaned up, or removed from the cell. This process can be compared to burning coal for energy; energy is provided, but it is very bad for the environment. Respiration, on the other hand, could be compared to solar or wind energy. It takes the same product, pyruvate, and extracts energy from it in a way that is \'cleaner\' for the cell, and in turn generates more energy. The **Krebs cycle**, also called the citric acid cycle, is the second preparatory step for cellular respiration in aerobic bacteria. The Krebs cycle also occurs in the cytoplasm of bacteria. In the Krebs cycle, pyruvate is broken down into carbon dioxide. In addition to generating ATP, the Krebs cycle and glycolysis generate hydrogen ions (H+), and electrons as shown in this figure. The electrons and H+ are harvested in cellular respiration to generate energy on the electron transport chain. -----------------------------------------  ----------------------------------------- **The Kreb cycle** The Kreb cycle is described a cycle because the substance at the end of the sequence is the same as the substance at the beginning. All the reactions are catalysed by enzymes, and this happens in the cell membrane of bacteria. Before pyruvic acid molecule enter the Kreb cycle, it undergoes a change, an enzyme removes a carbon atom from the pyruvic acid molecule and release the carbon as carbon dioxide (CO~2~). The remaining two carbon atoms are combined by coenzyme A to form acetyl-coenzyme or simple acetyl-CoA which then enters the Kreb cycle. The process of producing Acetyl Co-A from pyruvate through glycolysis is called pyruvate oxidation. Acetyl-CoA combines with the 4 carbon oxalocetic acid to citric acid, which is a six-carbon acid. Citric acid is converted to alphaketoglutaric acid which has only 5 carbon atoms. The sixth carbon is lost as CO~2.~ Succinic acid is then converted to fumaric acid, which is the converted to malic acid and malic acid to oxaloacetic acid. The cycle is now complete and oxaloacetic acid is ready to unite with another molecule of acetyl-CoA. **Krebs Cycle Products (x2)** 1. Three molecules of nicotinamide adenine dinucleotide (NADH): transfer their electrons to the next step of the pathway, the electron transport chain, which produces ATP through oxidative phosphorylation. 2. One molecule of flavin adenine dinucleotide (FADH2): transfers its electrons to the next step of the pathway, the electron transport chain, which produces ATP through oxidative phosphorylation. 3. One ATP/GTP: used for energy and the functioning of further steps of the pathway. 4. Two molecules of CO2: used for exhalation. How Does Stage 2 of Cellular Respiration Benefit a Cell? **Oxidative Phosphorylation** Oxidative phosphorylation refers to a sequence of reactions in which two events happen. Pairs of electrons are passed from one chemical substance to another, and the energy released during the process is used to combine phosphate with ADP to form ATP. Thirty four out of 38 molecules of ATP produced during glucose catabolism is manufactures here and it takes place in the cell membrane of the bacteria. Two important coenzymes that function in oxidative phosphorylation are NAD and FAD. Another important molecule are the cytochromes. Cyto chromes are a set of protein cellular pigments that contain iron ions that accept and release electrons. Because oxidative phosphorylation involves the passage of electrons, it is often called electron transport chain. The actual mechanism for ATP formation is called chemiosmosis but it involves both chemical and transport (osmosis) process. What happens in chemiosmosis? A. Coming from the glycolysis or Kreb cycle, a conenzyme (eg., NADH or FADH) transports electron pairs to the cytochrome in the cell membrane B. The NAD or FAD coenzymes is regenerated for reuse. C. As the cytochromes transport the electron pairs among themselves, they release energy, which fuels the transport of protons across the cell membrane at three points. D. Each set of protons the re-enters the cytoplasm pf the cell through a protein channel lined with ATP synthetase. E. An ADP molecule is joined with a phosphate each time a set of protons move through the channels, thereby accounting for three molecules of ATP molecules produced for each electron pair. F. The electron combines with other protons to form water molecules. Chemiosmosis occurs only in the structurally intact membrane. If the membrane is damaged, proton movement cannot take place, the synthesis of ATP stops even though electron transport through the cytochrome system continues. With the end of ATP production, the organism dies. This is principle behind the activity of some antibiotics and disinfectants.  **Figure 2: Chemiosmosis in bacteria** Energy yield in aerobic respiration can be summarised as follows; 6ATPs from the reaction in glycolysis via 2 NADHs 2ATPs from the net gain in glycolysis 6 ATPs from preparation of the Krebs cycle 2 ATPs from the reaction (D) in the Kreb cycle via GTP 18 ATPs from 6NADH from the Kreb cycle 4 ATPs from 2 FADH~2~ from the Kreb cycle The total number of ATP id 38 molecules of ATP. AT 7300 molecules of ATP, this yields 277,400 calories of energy preserves from the energy in glucose. It also completes the equation for aerobic respiration: C~6~H~12~O~6~ + 6O~2~ + 38ADP →6CO~2~ +6H~2~O + 38ATP A picture containing text, font, screenshot, design Description automatically generated Figure 3: Total yield of energy from aerobic respiration **Catabolism for other carbohydrates** Each carbohydrate utilises a different pathway for its catabolism. Sucrose is first digested by the enzyme sucrase into 2 different molecules, glucose, and fructose. The glucose molecule enters the glycolytic pathway directly, but the fructose molecule is first converted to fructose-1-phosphate. Fructose-1-phosphate undergoes further conversions and a molecule split before it enters glycolytic pathway as dihydroxyacetone phosphate (DHAP). Another sugar lactose which is a disaccharide is broken into two by the enzyme lactase to glucose and galactose. Glucose enters the pathway, but galactose goes a number of changes before it enters the glycolytic pathway as glucose-6-phosphate. Polysaccharides undergo a system of changes before entering the mainstream glycolysis. Starch and glycogen are metabolised as enzymes remove one glucose at a time and convert it to glucose-6-phosphate for entry into glycolysis. **Catabolism of Protein and fats** Although proteins are not considered energy sources, cells utilise them for energy when carbohydrates and fats are in short supply. Fats are extremely valuable as energy source because their chemical bond contain enormous amounts of chemical energy. For protein to enter the glycolytic pathway, they are first broken down to amino acids. By **deamination,** the amino group in the amino acids are removed and replaced by a carboxyl group to form components normally occurring in the carbohydrate. For example, alanine is converted to pyruvic acid and aspartic acid is converted to oxaloacetic acid. Fats consist of fatty acids bonded to a glycerol molecule. In preparation for catabolism, the fatty acids are separated from the glycerol by enzyme lipase. The glycerol portion is then converted to DHAP. The fatty acids go through a series of conversions under **beta oxidation** where enzymes convert each unit to a molecule of acetyl-CoA ready for the Kreb cycle. Each turn of the Kreb cycle yields 14 molecules of ATP, so from 16 carbon fatty acids (eight 2-carbon units), there is a substantial energy yield. **Anaerobic respiration** Bacteria that metabolise carbohydrate through anaerobic respiration do not use oxygen as the electro acceptor. Even without oxygen the electron transport takes place by oxidative phosphorylation. Instead of oxygen, anaerobic bacteria use an inorganic molecule as a final electron acceptor. For example, *Escherichia coli* uses nitrate ions (NO~3~^-^) at the end of the cytochrome chain. This is helpful to microbiologists in the identification of nitrite producers. Electrons combine with the nitrate ions and convert it to nitrite ions. Members of the genus *Desulfovibrio* used sulphate ions (SO~4~^-^) for anaerobic respiration. The sulphate combines with electrons and changes it to hydrogen sulphide. The archaebacteria use carbon dioxide as their final electron acceptor to produce large amounts of methane (CH~4~). **Fermentation** Fermentation is a type of anaerobic process because it does not use oxygen as a final electron acceptor instead it uses an organic compound usually an intermediary in the metabolic pathway. For example, the bacterium *Streptococcus lactis* practices fermentation by using pyruvic acid to accept electrons and protons from NADH. In the yeasts eg *Saccharomyces,* pyruvic acid is first converted to acetaldehyde, a process that involves the release of CO~2~ (This step is important in bread making). Acetaldehyde then serves as an electron acceptor for the electrons and protons of NADH and the acetaldehyde converts to ethyl alcohol. The liquor industry used ethyl alcohol to make alcoholic beverages such as beer and wine. **Read up chemosynthesis.**