Microbial Metabolism PDF

**[Metabolism/Energy Flow Student Notes]{.smallcaps}** ====================================================== [I. The Basics]{.smallcaps} [A. Metabolism = all chemical reactions in a cell, both catabolic and anabolic reactions]{.smallcaps} -**Catabolic reactions** = degradative reactions: large s...

**[Metabolism/Energy Flow Student Notes]{.smallcaps}** ====================================================== [I. The Basics]{.smallcaps} [A. Metabolism = all chemical reactions in a cell, both catabolic and anabolic reactions]{.smallcaps} -**Catabolic reactions** = degradative reactions: large substances are broken down into smaller substances to release energy and to supply building blocks for making macromolecules -Energy is captured in ATP/other high-energy molecules (PEP)/proton gradients; cells require energy for biosynthesis (anabolism), motility, active transport **-Anabolic reactions** = biosynthetic reactions: smaller substances are joined together using energy to synthesize larger substances (macromolecules) for cell structure/products (enzymes, organelles, etc) -**ATP** (adenosine triphosphate) is the major energy supplier; it links anabolic and catabolic reactions [B. Energy capture via electron transfer]{.smallcaps} 1\) Electrons carry energy and can transfer energy from one molecule to another 2\) **Electrons can be transferred as naked electrons or as part of hydrogen atoms (H = H^+^ + e^-^)**, therefore hydrogen atom transfer represents electron transfer 3\) Electron transfer is involved in ATP synthesis 4\) **Types of electron transfer: oxidation** = loss of electrons to an electron acceptor; **reduction** = gain of electrons from an electron donor; **redox reactions** = one substance loses electrons and another substance accepts those electrons -A substance ***losing electrons*** is **oxidized** and is called an ***electron donor or a reducing agent*** *-A* substance ***gaining electrons*** is **reduced** and is called an ***electron acceptor or an oxidizing agent*** -Redox reactions frequently involve **electron/H carriers (NAD+, NADP, FAD**) -**OIL RIG**: Oxidation Is Losing electrons Reduction Is Gaining electrons [C. Enzymes]{.smallcaps} 1\) Metabolic reactions in cells require **enzymes because:** a) metabolic reactions occur too slowly in cells to sustain life (T too low); and b) increasing temperature to increase reaction rates would kill cells (denature proteins/nucleic acids) **2) Enzymes = protein catalysts or RNA (ribozyme) catalysts that** increase reaction rates by lowering the activation energy (energy required to start the reaction) of chemical reactions -The amino acid sequence of an enzyme dictates its folding and 3-dimensional shape = functional shape; denaturation (heat, pH) destroys 3-D structure -3-D structure creates an **active site** where the enzyme binds its *specific* **substrate (= substance the enzyme binds to and works on)** -Enzyme (E) and substrate (S) bind and form enzyme-substrate (ES) complexes → bonds of substrate are rearranged → product (P) is formed → product is released from active site → active site is free to bind more substrate; enzyme is not consumed and can be used over and over \- E + S → E:S → E + P -Enzymes are substrate specific \[act on a single substrate or closely related (shape, charge, size) substrates\] and generally catalyze only a single reaction -Enzyme suffix = -***ase***; enzymes are named according to substrate and/or function ex. phosphofructokinase = enzyme which adds phosphate to fructose 6-phosphate -Enzyme activity can be regulated by: competitive inhibition; non-competitive inhibition; feedback inhibition **3) Coenzymes and Cofactors** = substances required by some enzymes for full activity -Enzyme without cofactor/coenzyme = apoenzyme; with cofactor/coenzyme = **holoenzyme** -**Coenzymes** = organic molecules (NAD+, FAD, cytochromes), loosely associated with enzyme; often formed from vitamins (niacin → NAD+) -**Cofactors** = inorganic molecules; eg., magnesium, calcium, zinc; often improve fit of substrate in active site **4) Metabolic Pathways** = series of chemical reactions where the product of the first reaction is the substrate for subsequent enzyme-catalyzed steps -Each step in a metabolic pathway requires a specific enzyme: pathway A → B→ C → D → E (endproduct) -If one enzyme in a pathway is missing or defective, the cell cannot form the final product of the pathway nor any of the intermediates downstream from the missing enzyme = metabolic diseases eg., phenylketonuria (aspartame is toxic for phenylketonurics) [II. Carbohydrate metabolism and energy production in chemoheterotrophs]{.smallcaps} [A. Aerobic Respiration]{.smallcaps} 1\) Oxidation (removal of electrons) of glucose (a reduced molecule) releases energy that can be used for ATP synthesis and ion gradient formation. These sources of potential energy are then used to do cellular work: biosynthesis/anabolism, active transport, motility **2) Glycolysis** = first step in energy release; one molecule of glucose (C6) is broken into 2 molecules of pyruvate (C3) ; 2 ATP are synthesized via substrate level phosphorylation and 2 NADH are produced; net 2 ATP (2 ATP required to fuel the first reaction) -**Substrate level phosphorylation** = the formation of ATP by directly transferring a phosphate group to ADP from an intermediate substrate during catabolism **-The NADH used must be regenerated because the cell's supply of NAD+ is finite; using it up will result in a shut-down of glycolysis** **3) *Tricarboxylic Acid Cycle=Krebs Cycle=Citric Acid Cycle*** -***The TCA cycle will turn twice per glucose molecule; GTP (ATP) will be produced by substrate level phosphorylation*** Big Picture: 2 pyruvate (C3) become 2 acetyl CoA (C2 + CoA) and are oxidized in a cyclic manner, generating 2 ATP (GTP), 6 NADH and 2 FADH~2~; FADH~2~ will generate ATP via ETC, NADH will generate ATP via ETC -For every turn of the cycle, 2 carbon molecules are released as CO~2;~ oxaloacetic acid (C4) is regenerated and can combine with another acetyl CoA to continue the cycle -The Krebs Cycle results in the oxidation (removal of electrons = H atoms) of glucose -The Krebs Cycle is used primarily by aerobes although many anaerobes retain the enzymes and use the intermediates in synthetic pathways **4) Electron Transport Chain**: respiration, chemiosmosis and ATP synthesis -Two functions: to accept electrons from electron carriers and transfer them to other electron acceptors; and to use energy released from electron transfer to **create a proton gradient across membrane (source of potential energy) → "proton motive force"** a\) **Electron carriers** are located in cell membrane of prokaryotes and in inner mitochondrial membrane of eukaryotes -**Cytochromes** = proteins containing *porphyrin rings complexed with iron, eg., cytochrome C, chlorophyll in plants* *-**Flavoproteins** = proteins containing flavin (from riboflavin = vitamin B~2~)* *-**Ubiquinones** = ubiquitous lipid-soluble non-protein electron carriers derived from Vitamin K, eg., Coenzyme Q* -**Metal-containing proteins** = proteins complexed to metal ions (Fe, S, Cu) eg., iron-sulfur proteins **b)** Some carriers carry only protons while others carry only electrons; result: H+ gets pumped across the membrane, creating a proton gradient (higher concentration of protons on the outside of the membrane than on the inside) c\) **Aerobic respiration: terminal electron acceptor = inorganic O** **2 e^-^ + 2H^+^ +1/2 O~2~ = H~2~O** d\) **Chemiosmosis**: an electrochemical gradient across a membrane is used to perform cellular work, eg., drive ATP synthesis via ATP synthase in membrane -Eventually, the proton concentration on the outside of the membrane becomes to great, and the protons flow back through membrane via ATP synthase; this produces the energy used to phosphorylate ADP to produce ATP -**Oxidative phosphorylation** = the production of ATP using energy derived from redox reactions of an ETC **-Big picture of aerobic respiration**: high energy electrons of glucose are stripped away during glycolysis and the Krebs Cycle, and then carried by NADH and FADH~2~ to the cell membrane where ETC is located, passed down ETC, and lose energy as the energy is used to pump protons across cell membrane creating a PROTON GRADIENT. The proton gradient is used to drive ATP SYNTHESIS via ATP SYNTHASE. **e) Summmary of aerobic respiration**: **C~6~H~12~O~6~ + 6O~2~ → 6CO~2~ + 6H~2~O + (36- 38) ATP + heat** **f) Anaerobic respiration: (prokaryotes only)**: respiration using molecule other than O~2~ as the terminal electron acceptor at end of ETC -Example: NO~3~^-^ (nitrate) can be used as a terminal electron acceptor -Up to 36 ATP/glucose can be generated [B. Fermentation]{.smallcaps} -Occurs in the absence oxygen or when the cell lacks an electron transport chain; serves to oxidize the NADH produced during glycolysis -To regenerate NAD from NADH, the electrons of NADH (H + e^-^) are passed to an organic molecule, ie., an organic molecule is the terminal electron acceptor -There are **many *different*** ***fermentation pathways*** involving ***different enzymes*** to produce **many *different products; the name of a pathway is often based on the products (see pp743-747 in the Bauman text to learn more)*** ***-In general, an organism has enzymes for one fermentation pathway only, eg., yeast only have enzymes for the alcoholic fermentation*** -**Fermentation pathways and products can be used for diagnostic purposes**, eg., *E. coli* O157:H7 cannot ferment sorbitol whereas other strains of *E. coli* can; lactic acid bacteria (*Streptococcus*) ferment glucose into lactic acid; clostridia use the butyric acid (found in rancid butter, parmesan cheese, vomit; characteristic odor of gangrene) fermentation; *Enterobacter* and *Klebsiella* use the butane-diol fermentation (detected by the Voges-Proskauer test component of the IMViC tests used in clinical diagnosis) -**Advantages**: NADH regenerated to NAD+, and tasty end-products (yogurt, cheese, soy sauce, sauerkraut, beer, wine) -**Disadvantages**: end-products are incompletely oxidized organic molecules which still contain a lot of energy; the end-products are frequently toxic (eg., acids, alcohols) -The early Earth atmosphere was anaerobic; fermentation evolved as a non-oxygen-requiring means of regenerating NADH to NAD^+^ -The evolution of porphyrins (see above) lead to the evolution of chlorophyll and cytochromes, allowing for respiration and the production of oxygen by primitive cyanobacteria to give rise to our current oxygen-rich (oxidizing) atmosphere [C. Glycolysis Alternatives]{.smallcaps} **1. The Pentose Phosphate Pathway** -Primarily a pathway for using glucose to produce 5C sugars (pentoses) used for nucleic acid synthesis (ribose), and other anabolic pathways -Produces 1 ATP/glucose + 2 NADPH (needed for synthesizing DNA nucleotides, steroids, and fatty acids) -G6PD deficiency causes favaism but protects against malaria **2. The Entner Doudoroff Pathway** -Catabolizes glucose to pyruvic acid using different enzymes than glycolysis -Produces 1 ATP/glucose + NADPH + G3P (enters Krebs Cycle) -Used by *Pseudomonas aeruginosa* and *Enterococus faecalis;* can be used diagnostically by assay for 2-keto-3-deoxy-6-phosphogluconic acid (unique intermediate) III. **Comparison of Fermentation and Aerobic Respiration** **Oxygen Required** **ATP Produced** -------------------------- --------------------- ---------------------- **Fermentation** No 2 ATP/glucose **Aerobic respiration** Yes \~38 ATP/glucose **Anerobic respiration** No Up to 36 ATP/glucose [III. Photosynthesis ]{.smallcaps} **-The process by which photoautotrophs (photo = light + auto = self + troph = food/nourishment; "self-feeding from light") synthesize their own food using energy from light; green plants and cyanobacteria = photoautotrophs** -Chlorophyll a of the chloroplast thyllakoid photocenter converts light energy into chemical energy to produce glucose (food) and oxygen: 6CO~2~ + 6H~2~O + *light* → C~6~H~12~O~6~+ 6O~2~ **-This occurs in two series of reactions: a) light reactions (cyclic and non-cyclic photophosphorylation) in which energy is captured in photocenters (light excites chlorophyll electrons which pass down an ETC to generate ATP); and b) light-independent ("dark") reactions in which CO2 is reduced (H^+^ + e^-^ are added) in the Calvin-Benson Cycle** **-Cyclic photophosphorylation occurs in photosystem I; all photosynthetic organisms have PS I** **-Non-cyclic photophosphorylation uses both PS I and PS II to generate ATP, NADPH, and O~2~.** **-Green plants, algae (plant-like protists), and cyanobacteria have PS II which is required for oxygenic photosynthesis** **-Calvin-Benson Cycle: 3CO~2~ + 3 ribulose bisphosphate (C5) +6 ATP + 6 NADPH → 6 G3P → glucose + 5 G3P (continues cycle)** -The organic molecules photoautotrophs produce are essential for the survival of ***chemoheterotrophs*** (chemo = chemical + hetero = other + troph = food; "feeding from organic molecules produced by others") -Chemoheterotrophs are organisms (such as ourselves) which require pre-formed organic molecules as carbon and energy sources **Photoautotrophs** **Chemoheterotrophs** -------------------------------------------------------------------------------------------- -------------------------------------------------------------------------------------------------------------- Have chlorophyll or other photosynthetic pigments (bacteriochlorophyll, bacteriorhodopsin) Lack chlorophyll or other photosynthetic pigments Green plants, cyanobacteria, other photosynthetic prokaryotes Humans, animals, fungi, protozoa, non-photosynthetic prokaryotes; most pathogens (bacteria, fungi, protozoa) [IV. Lipid Catabolism]{.smallcaps} -Fatty acids are catabolized via beta-oxidation (see below); 14-17 ATP are produced per cycle of beta-oxidation [V. Protein Catabolism]{.smallcaps} -Proteins are cleaved into individual amino acids by proteases, the amino (NH~2~) groups are removed by deamination (deaminases), the carboxyl groups are removed by decarboxylation (decarboxylases), and the R groups are removed by various enzymes. The resulting C2 molecules are routed to the Krebs cycle. -Maple syrup urine disease results from defect(s) in the enzymes involved in metabolizing branched-chain amino acids (leucine, isoleucine, valine) References: Bauman, R. *Microbiology: with diseases by taxonomy*, 2^nd^ edition, 2007. Pearson Education, San Franscisco, CA. Black, J. G. *Microbiology: principles and practices*, 6^th^ edition, 2005. John Wiley & Sons, Inc., Danvers, MA. Carberry-Goh, K. "Metabolism Review", Summer 2005. Garrett, R. H. and Grisham, C. M. *Interactive Biochemistry*: , \[webpage\]. Accessed September 30, 2006. Todar, K. *Todar's Online Textbook of Bacteriology*, , \[webpage\]. Accessed September 30, 2006. [Study Guide]{.smallcaps} 1\. **Metabolism**: what is metabolism; what is anabolism; what is catabolism; what are the goals of metabolism? 2\. **Metabolism**: what is oxidation; what is reduction (OIL RIG); what is a REDOX reaction; what is a metabolic pathway; why does metabolism occur in a step-wise manner? 3\. **Enzymes**: what are enzymes; what is their function; what are their components; how are they named; how do they work; what is the name of the enzyme that's involved in the first stage of glycolysis, the pentose phosphate pathway, and the Entner-Doudouroff pathway? 4\. **Glycolysis**: what are the starting and end products of glycolysis; what are the stages of glycolysis; how many ATP's are produced per molecule of glycose; how many NADH's are produced; what is/are the purpose(s) of glycolysis; where does glycolysis occur in the cell; does glycolysis require oxygen? 5\. **The Pentose Phosphate Pathway**: what is the starting molecule; what is the primary purpose of this pathway? -What is the enzyme that if mutated can cause hemolytic anemia in humans? What can trigger episodes of hemolytic anemia in people who have the mutation? What is the advantage of having this mutation? 6\. **The Entner-Doudouroff Pathway**: what is the starting molecule; what is the intermediate produced by this pathway that can be used for diagnosing bacterial infections; what is this pathway's main purpose? 7\. **The Krebs Cycle**: what must be done to pyruvate for it to enter the Krebs Cycle; what are the starting and end-products; how many ATP's are produced per turn, per glucose; how many NADH's are produced per turn, per glucose; what is its purpose? 8\. **Electron Transport**: what is an electron transport chain; what are its components/carrier molecules; what is chemiosmosis; what is proton motive force and what does it do; what are the starting and end products; what is the terminal electron acceptor of an aerobic ETC; what is used as the terminal electron acceptor of an anaerobic ETC; how many ATP's are made using each type of ETC; what is the purpose of an ETC? -Can an organism have more than one ETC? Why would it want to? 9\. **Fermentation**: what is fermentation; what is its starting molecule; what are some of its end-products; what is its purpose? -How was fermentation important in recent (post-WW II) history? 10\. **Other catabolic pathways**: how are proteins catabolized; how are fats catabolized; how many ATP's are generated per cycle of β-oxidation; why is it called β-oxidation? 11\. **Photosynthesis**: what are the light reactions, and what are the products; what are photocenters and what is chlorophyll; what are the dark reactions and what are the products; how many CO~2~, ATP, and NADPH does it take to make a molecule of glucose; what is the key enzyme of the Calvin-Benson cycle; what is the importance of photosynthesis? -Which photocenter is responsible for oxidative photosynthesis? Which photocenter do all photosynthetic organisms have? What is the importance of oxidative photosynthesis? -What is cyclic photophosphorylation, and how does it work? What is non-cyclic photophosphorylation and how does it work? 12\. **Biosynthetic pathways**: what is an amphibolic pathway; can some pathways be both anabolic (synthetic) and catabolic (degradative)?