lecture 8 metabolism
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lecture 8 metabolism

Course Number: BSCI 223, Spring 2011

College/University: Maryland

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Martian meteorite may have held life Feb. 10 2006, NewScientist.com news service Kimm Groshong A mix of carbon compounds filling the miniscule veins in a Martian meteorite has refuelled the debate on the possibility of life on Mars. Similarities between the carbon-rich filler and that found in fractured volcanic samples from the Earth's ocean floor dangle the possibility that life produced the Martian material,...

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meteorite Martian may have held life Feb. 10 2006, NewScientist.com news service Kimm Groshong A mix of carbon compounds filling the miniscule veins in a Martian meteorite has refuelled the debate on the possibility of life on Mars. Similarities between the carbon-rich filler and that found in fractured volcanic samples from the Earth's ocean floor dangle the possibility that life produced the Martian material, say scientists. A team of researchers led by David McKay and Everett Gibson of the Johnson Space Center in Houston, US, raise the scenario as just one possibility after extensively analysing new samples of the Nakhla meteorite. The UKs Natural History Museum recently provided the team with fresh samples from the interior of the meteorite, which broke into many pieces upon landing in Egypt in 1911. Near the tube-like veins in the rock, researchers found iddingsite a mineral also formed on Earth, mainly through alteration of an iron-based mineral called olivine by water. And within the cracks, they found carbon-rich material that appears dark brown or black. "Indigenous stuff" Astrobiologists look for carbon and water in their search for extraterrestrial life. Carbon is the building block of terrestrial life, forming the basis for organic chemistry, and water is necessary to support all forms of life on Earth. The team outlined possible sources of the carbon-containing components. Either a carbon-bearing impactor introduced them to Mars between 600,000 and 700,000 years ago or they are "products of biogenic activity and introduced by groundwater into the fracture features in Nakhla. Enzymes convert substrates to products reactions that make up steps in metabolic pathways Enzymes are catalysts that decrease activation energy of a reaction, bringing reactants together at physiological temperatures Enzymes often require cofactors or coenzymes. Coenzymes serve to transfer functional groups from one reaction to another, for example NAD is a coenzyme that transfers electrons. Enzymes control whether or not reactions occur. Enzyme activity can be regulated via binding of a regulatory molecule to the enzyme allosteric site. Some enzymes have allosteric sites. In Feedback inhibition the end product of the pathway serves as a regulator to an enzyme early in the pathway. When the endproduct is present the endproduct binds to the allosteric site, inhibiting the enzyme from binding to the substrate. The metabolic pathway cannot proceed past this reaction. Reactions that generate energy are coupled to the phosphorylation of ATP. This is substrate level phosphorylation. The energy is stored in the high energy phosphate-phosphate bond Oxidation reactions are half reactions, always coupled to reduction reactions. Oxidation of a subsrate may be coupled to reduction of NAD. NAD is reduced to NADH. This coenzyme is a carrier of electrons. The electrons are eventually transferred to a terminal electron acceptor. The NADH is oxidized and the terminal electron acceptor is reduced. The most electron loving electron acceptor is oxygen. This is the terminal electron acceptor in aerobic respiration. E. coli is the most studied Bacterium. Much of what is reported in General Microbiology texts books as representative for bacteria is describing the example of what is happening in E. coli. E. coli is a chemoorganoheterotroph. E. coli uses glucose efficiently as a carbon and energy source. Most efficient oxidation of glucose occurs via aerobic respiration. First step of aerobic respiration with glucose as a carbon source is transport of glucose. As E. coli is gram negative, glucose passes through outer membrane via porins and then through innner membrane via group translocation. Group translocation requires 1 ATP and phosphorylates glucose to G-6-P (first step in gylcolysis). Gylcolysis can be used to generate energy. With glucose as the substrate, this molecule is oxidized to two pyruvate molecules. Follow the Carbon backbone, ATP and electron flow to NAD to understand the pathway. Pyruvate is further oxidized via transition to the TCA cycle. At the completion of this cycle the carbon backbone is completed broken down (catabolism) to 6 CO2 molecules. Energy has been generated and stored in ATP, electrons remain in the reduced form of the NADH coenzyme. These must be passed to an electron acceptor. In the electron transport chain the electrons from oxidation of glucose (associated with NADH) are transferrred to the terminal electron acceptor. In aerobic respiration this will be oxygen, anaerobic respiration this will be another inorganic molecule such as nitrate. If viable count declines but total cell count remains constant, what phase of growth is a culture in? Log (number of viable cells) 1. 9% 2. 19% 3. 26% 4. 22% 5. 24% lag phase exponential phase Time stationary phase death phase You cannot tell from the information given 8.2 Enzymes are 1. 24% 2. 18% 3. 29% 4. 20% 5. 10% Encoded by genes Protein in nature Reduce the activation energy of a reaction Two of the above answers are correct All answers are correct Complex vs minimal media What do bacteria need to grow? What is in media? What components in medium 1 make it complex? Medium #1 5 g yeast extract 20 g tryptone extract 0.5 g NaCl g 3.6 glucose 1 l H20 18% 7% 22% 32% 13% 9% Medium #2 10.5 g K2HPO4 4.5 g KH2PO4 1 g MgSO4 10 g Polyurethane 1 l H2 0 1. 2. 3. 4. 5. 6. Yeast extract Tryptone NaCl Glucose Water More than one component Which components of medium # 1 could be added to medium # 2 and still leave it defined? Medium #1 5 g yeast extract 20 g tryptone extract 0.5 g NaCl 3.6 g glucose 1 l H20 15% 15% 23% 18% 16% 13% Medium #2 10.5 g K2HPO4 4.5 g KH2PO4 1 g MgSO4 10 g Polyurethane 1 l H2 0 1. 2. 3. 4. 5. 6. Yeast extract Tryptone NaCl Glucose Water More than one component For ETPUM growth Medium #1 is rich and complex Medium #2 is minimal and defined Medium #1 5 g yeast extract 20 g tryptone extract 0.5 g NaCl 3.6 g glucose 1 l H20 Medium #2 10.5 g K2HPO4 4.5 g KH2PO4 1 g MgSO4 10 g Polyurethane 1 l H20 You are excited because, in Medium #2, ETPUM utilizes polyurethane as its energy source and its sole source of carbon and nitrogen, a finding that raises the possibility that ETPUM could be a useful tool for bioremediation of polyurethanecontaining wastes (in landfills, etc.). You have also made some progress in characterizing the central metabolic pathways and related biochemical activities of ETPUM. In particular, you have discovered that: ETPUM secretes an enzyme (polyurethanase) that catalyzes degradation of polyurethane, generating citric acid (citrate) as a product the cytoplasmic membrane of ETPUM contains an ABC transport system capable of transporting citrate across the membrane at the expense of 4 ATP molecules (hydrolyzed to form ADP and phosphate) per molecule of citrate transported the cytoplasm of this organism contains all of the enzymes required for glycolysis, and for the TCA cycle the cytoplasmic membrane of ETPUM contains proteins that form a functional electron-transport pathway (that uses O2 as the terminal electron acceptor) Given that polyurethane is a huge polymer (MW >>100,000 Daltons), why is it important that the polyurethanase is a secreted enzyme? If we assume that the polyurethane is the source of energy for the organism, how can material (carbon atoms) from it find its way into the central metabolic pathways of this microbe? What is the entry point? What happens after its entry into the metabolic pathway? NOTE: This figure has been modified from Book figure Net Gain 2 ATP Fermentation Regeneration of NAD+, No additional ATP generated Net gain from fermentation of glucose, 2 ATP Howisreducingpower(NADH)converted intoATP? Processiscalledrespiration =OxidativePhosphorylation UtilizeselectrontransportchainandATP synthase Aerobic Respiration Pathway Carbon Glucose Glycolysis 2Pyruvate Prep step Krebs Cycle 2Acetyl CoA + 2CO2 4CO2 Electrons Energy 2NADH 2NADH 6NADH 2FADH2 2ATP 2ATP Chemiosmotic generation of ATP (10 NADH X 3) + (2 FADH2 X 2) = 34 ATP 38 ATP How could Polyurethane work as a carbon source? ABC transporter How could Polyurethane work as a carbon source? Group translocation (phosphotransferase system) precursor metabolites Citrate transport TCA precursor metabolites What about energy? How can ETPUM get enough ATP using citrate as C/Energy source? (-4 ATP) Citrate transport TCA (+1 ATP) How many ATP will ETPUM produce from aerobic respiration when using citrate as a carbon source? 13% 13% 22% 22% 19% 12% 1. 2. 3. 4. 5. 6. 2 ATP 8 ATP 11 ATP 32 ATP 34 ATP 38 ATP 9 2 (-4 ATP) (+1 ATP) = Total 8 ATP PAK 1.3 Medium #1 5 g yeast extract 20 g tryptone extract 0.5 g NaCl 3.6 g glucose 1 l H20 Medium #2 10.5 g K2HPO4 4.5 g KH2PO4 1 g MgSO4 10 g Polyurethane 1 l H2 0 Why doe thegrowth of EPTUM re s quireoxyge n? How can ce ge ratee rgy in theabse of O2? lls ne ne nce How m ATP can onege fromfe e uch t rm ntation? C onefe e citrate an rm nt ? Why is oxygen required? Entry point - 4 ATP for transport into cell + 1 ATP 3 NADH produces 9 ATP 1 FADH produces 2 ATP + 11 ATP 12 ATP- 4 ATP = 8 ATP How can cells generate energy in the absence of O2? 16% 23% 23% 24% 15% 1. 2. 3. 4. 5. Fermentation Anaerobic respiration Oxidative phosphorylation Group translocation More than one answer is correct PAK 1.3 Medium #1 Medium #2 5 g yeast extract 10.5 g K2HPO4 20 g tryptone extract 4.5 g KH2PO4 0.5 g NaCl 1 g MgSO4 3.6 g glucose 10 g Polyurethane 1 l H20 1 l H2 0 Why doe thegrowth of EPTUM re s quireoxyge n? How can ce ge ratee rgy in theabse of O2? lls ne ne nce HowmuchATPcanonegetfromfermentation? Can EPUTUM ferment Citrate? 42% 58% 1. Yes 2. No Which components of medium # 1 could be used as an energy source? Medium #1 Medium #2 5 g yeast extract 10.5 g K2HPO4 20 g tryptone extract 4.5 g KH2PO4 0.5 g NaCl 1 g MgSO4 3.6 g glucose 10 g Polyurethane 1 l H20 1 l H2 0 6% 20% 12% 27% 16% 20% 1. 2. 3. 4. 5. 6. Yeast extract Tryptone NaCl Glucose Water More than one component Hydrolysis Hydrolysis Terminology Energy produced (ATP yield) Aerobic Respiration breakdown of nutrients to produce energy using electron transport chain with O2 as final electron acceptorsubstrate fully oxidized Anaerobic Respirationbreakdown of nutrients to produce energy using electron transport chain with S04, N03 or others as final electron acceptor substrate fully oxidized Fermentationbreakdown of nutrients to produce energy and recycles NAD+--substrate partially oxidized

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