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Microbial Physiology and Biochemistry

Part 1: Enzymes, intro to Energetics, and Membrane transport

This portion of the course provides the details behind how microorganisms obtain energy and grow. The act of growth requires the conversion of chemical or light energy to biochemical energy that the organism can use. There are relatively few ways that chemical energy can be captured by living organisms and can be used to do work, such as building new cell material. What you will be learning are mostly all variations on the same theme. The theme is the coupling of electron transfer reactions from the breakdown of organic or inorganic compounds (catabolism) to the production of energy or cellular material (anabolism). Collectively the study can be called microbial metabolism. In order to couple chemical reactions to energy production a living system must have a way of controlling these reactions. You will want to review your thermodynamics because I use thermodynamic principles in the explanations. In most cases, although the free energy change of the reactions are negative, indicating that energy will be released during the reactions, these reactions are not spontaneous, that is they have a high activation energy. This must be overcome by the living system in order to release the energy. This is accomplished by the use of enzymes. Enzymes Enzyme - a biological catalyst, composed of protein. characteristics increase rate of reaction does not effect thermodynamics (can catalyze both exergonic and endergonic

reactions) highly specific not used up - recycled Enzyme function - substrate attaches to enzyme at active site reaction takes place enzyme is released from product Mechanisms 1. alters geometry of substrate molecule slightly to enable reaction to proceed 2. holds substrates in proper orientation 3. concentrate substrates from dilute surroundings Nomenclature name ends in ase usually descriptive of the type of reaction and substrate e.g. substrate oxidase - enzyme that oxidizes substrate substrate hydroxylase - enzyme that hydroxylates substrate some names you just have to remember amylase - starch degrading enzyme

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cellulase - cellulase degrading enzyme Some enzymes contain additional non-protein groups e.g. heme (cytochromes) Coenzymes - bound loosely to the enzymes may associate with other enzymes serve as intermediate carriers of small molecules from one enzyme to

another Enzyme Diversity Microbial populations have the job of being the scavengers of the world. These organisms (fungi and bacteria) contain a breadth of enzymes capable of degrading any natural compound. Not every bacteria can degrade every compound, but for every compound that has evolved naturally, a population of bacteria will have also evolved capable of degrading the compound. The evolution of enzymes is a study of extreme interest to environmental engineers and microbiologists, as we are now asking the bacteria to metabolize compounds that are man made and have not been in the environment long enough (in some cases) for the organisms to become adapted to them. The study of enzyme evolution is in its infancy but it has been shown that in most cases a new enzyme will evolve from an old enzyme that performs the same reaction on a more natural chemical. For example if you think a compound would best be degraded by a specific type of reaction, then you should expose bacteria (or fungi) that can do the type of reaction you want to the new compound and hopefully sooner or later they will be able to use the new compound. This may take a long time, but in most cases it will work eventually. Many are trying to make designer enzymes, ones in which genetic engineering has been used to purposefully modify genes and create new enzymes. This may show great promise, some day, but a lot more work must be done to make the new enzymes stable in the cell. Organisms that are exposed to the compound over long periods of time and adapt to the compounds (enrichment) are mush more stable in the environment than genetically engineered microorganisms. Many biological reactions, especially in metabolism, involve the transfer of electrons therefore they can also be classed as redox reactions. This means that one compound is being reduced and the other is being oxidized. Redox Reactions See Chapter 2 from Rittman and McCarty or Appendix 1 in Brock Oxidation - loss of e-

e.g. Fe+2 Fe+3 + e- (half reaction) Reduction gain of electron e.g. 1/2 O2 + 2H+ + 2e- H2O (half reaction)

free electrons do not exist in nature so oxidation and reduction must be coupled.

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Balancing reactions 1. Balance electrons 2. Balance charge 3. Balance numbers of atoms on both sides e.g. Coupling nitrate reduction to glucose oxidation NO3- 1/2 N2 C6H12O6 6 CO2 electrons N from +5 0 5 e- are added glucose c = 0 CO2 (c = +4 x 6 = 24) 24 e- are lost. to balance multiply nitrate equation by 24/5 or 4.8 4.8 NO3- + C6H12O6 2.4 N2 + 6CO2 Charges are not balanced (use H+ to balance - charge or put H+ on the other side to bal +) 4.8 NO3- + 4.8 H+ + C6H12O6 2.4 N2 + 6CO2 Number of atoms is not balanced Use H2O to balance Os 4.8 NO3- + 4.8 H+ + C6H12O6 2.4 N2 + 6CO2 + 8.4 H2O Check charge 0 0 C 6 6 N 4.8 4.8 O 20.4 20.4 H 16.8 16.8 Redox Potential tendency of substances to accept electrons and become reduced written as reductions in table i.e. electrons on left Eo - reduction potential in volts at pH 7 Eo > 0 reaction favorable e.g. oxidize sulfide to sulfate with O2 4H2O + HS- SO4-2 + 8e- + 9H+ Eo = 0.217v

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4H+ + O2 + 4e- 2H2O Eo = 0.82v balance e- overall 2O2 + HS- SO4-2 + H+ Eo = 1.037v favourable can convert to G G = -n F Eo (where F is Faradays constant 96.48 kJ/volt; n is number of electrons) = -8 (96.48) (1.037) = -800 kJ/mol HS- or directly from free energy of formation Gf (kJ/mol) 2 O2 + HS- SO4-2 + H+

0 12.05 -744.6 -39.83 G = -796 kcal/mol HS- In order for a cell to gain energy from a reaction, the cell must get the compound into its interior so it can couple the chemical reaction to energy generation processes. As we have discussed previously, the membrane is a barrier to the entry of large molecules into the cell.

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TRANSPORT Most gasses and some small molecules can pass through the membrane but molecules larger than glycerol must be transported across the cell membrane. The outer membrane of Gram negative organisms contains embedded proteins called porins that allow most monomeric, and some dimeric compounds to pass through. A. Polymeric molecules Polymeric molecules such as proteins, cellulose and starch are too big to enter into the cell. So instead the cell exports enzymes that can break down the larger molecules into smaller units that can be transported into the cell. These enzymes are called extracellular enzymes and fall into two general categories. 1. Hydrolases - these enzymes add water across a bond (the opposite to the

condensation that produced the polymers) thus cleaving the bond and producing two new molecules. e.g.

protease - enzyme that cleaves the peptide bond of proteins resulting in the release of amino acids.

cellulase - enzyme that cleaves cellulose into glucose monomers amylase - enzyme that cleaves starch into glucose monomers chitinase - enzyme that degrades chitin 2. Peroxidases - these enzymes introduce a free radical into a molecule thus

causing it to be unstable. The compound usually undergoes some kind of bond cleavage as it becomes stable again. The enzymes require H2O2 (peroxide) for their activity. The enzymes are most often produced by fungi that grow on lignin (the woody material from plants) e.g. Phanerochaete chrysosporium. The enzymes are not very specific in activity and can act on a wide variety of substrates (usually aromatic)

These enzymes act outside the cell so they are not directly coupled to energy generation. The monomer products of the enzyme activity are then brought inside the cell where their metabolism can be coupled to energy generation. There are several different mechanisms in which a compound can enter into a cell. Some are more efficient than others and some require energy while others do not. The compounds are all brought into the cell by way of special transport proteins imbedded in the cell membrane. These proteins are very specific for specific compounds or groups of compounds.

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Transport Mechanisms 1. Diffusion The compounds simply diffuse through the membrane, this relies on the permeability of the compound and a concentration gradient. Compounds always diffuse from a high concentration to a low concentration. The more difference in concentration the faster the diffusion occurs. This requires no energy and is only applicable to gasses and some very small molecules. There are some diffusion coefficients in your text (transport section). 2. Facilitated diffusion specific proteins allow some of the larger compounds in relies on a concentration gradient important in outer membrane porins 3. Active transport Mechanisms by which the compounds are actively pumped across a membrane. All require energy of some sort All require specific protein carriers All are saturable Before we go on with this you must learn one more thing about the membrane. The membrane is not permeable to protons or hydroxyl radicals. During normal metabolism the cell pumps all protons out and keeps all hydroxyl ions in. What this does is sets up a battery type situation with protons and other positive ions on the outside and none on the inside. This creates a gradient of protons that make up a theoretical force that is used for work when a proton is allowed into the cell. The force has been called the proton motive force or PMF. It is composed of two separate smaller forces pH = the proton gradient = the charge gradient Pictorial representation of PMF

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This force is used for many things including transport. Types of active transport (simple ones) a) Symport two compounds entering or leaving together usually relies on the PMF H+ moves inside the cell therefore creating enough energy to allow

the other compound to be pulled in with it. We say that this is at the expense of pH b) Antiport one compounds comes in, the other goes out energy usually from pH again c) Uniport single compound entering or leaving cell usually only ions (+ can go in at expense of , - out) More complicated Active Transport d) Group translocation substance being transported is altered as it crosses the membrane example is the phosphoenolpyruvate dependent phosphotransferase

system. (you should all be familiar with this be now) Basically the sugar is phosphorylated as it crosses the membrane. There are several proteins involved. The phosphate is transferred from phosphoenolpyruvate to glucose through enzyme intermediaries. Text has an in depth explanation.

e) Binding Protein mediated transport This is found in Gram negative cells only. The Binding proteins (BPs) are located in the periplasm (space

between the two membranes) The proteins bind the compound and deliver it to the membrane (a

conformational change upon binding causes the protein to become hydrophobic and go towards the membrane.

Transport proteins in the membrane attract the BPs (hydrophobic interactions again)

This keeps the concentration of the compound in the periplasm low and allows the diffusion gradient across the outer membrane to remain strong.

The protein undergoes another conformational change when it gets to the transport protein and the compound is released to the transport protein. The BP goes back out for more.

The transport protein uses a high level ~P bond and transports the compound into the cell. The transport protein goes back to its original conformation and is ready to accept another compound.

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Microbial Energetics We have just discussed some specific ways in which the cell couples energy generating reactions to perform work that needs energy. These reactions require specific proteins and use a small number of compounds that can carry energy from one part of the cell to another. 1. Freely Diffusable Electron Carriers These molecules diffuse throughout the cell and participate in all oxidation reduction reactions either as electron acceptors or donors. i) NAD - nicotinamide adenine dinucleotide ii) NADP - NAD phosphate These molecules are also proton carriers NADH + H+ NAD+ + 2e- + 2H+ or NADPH + H+ NADP+ + 2e- + 2H+ These are most often involved in dehydrogenation reactions and carry electrons from the metabolic reactions to the electron transport chain for energy generation. The two molecules can be interconverted by an enzyme but usually take part in different types of reactions. NAD - usually carries electrons to electron transport NADP - usually carries electrons to biosynthetic pathways 2. Membrane Bound Carriers These are compounds that are bound in the membrane and are part of the electron transport chain. They accept electrons from the soluble carriers and pass them along from one to another until the electron is passed on to the terminal electron acceptor. You will get more on this later. 3. High Energy Phosphate Carriers ATP The best example is ATP. This molecule is the prime energy carrier of the cell. I like to call it the money of the cell. Cleavage of ATP to ADP generates energy and can be coupled to many reactions that require energy. ATP is not the highest energy carrier but it has a middle range so that it is not too hard to make. The P bond can be transferred from any one of the compounds above ATP to ATP and on down. ATP Production I - substrate level phosphorylation (SLP)

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transfer of a high energy phosphate bond from an organic compound to ADP to form ATP e.g. These reactions form part of the pathway for the metabolism of glucose. In the sequence an inorganic phosphate molecule is incorporated into an organic molecule and then transferred to ADP to form ATP

glyceraldehyde-3-P + Pi + NAD NADH + 1,3-diphosphoglycerate 1,3-diphosphoglycerate + ADP phosphoglycerate + ATP + NAD II - Conservation of High Energy Bonds In organisms that are fermentative in nature, there is very little energy

around. The organisms employ another type of high energy bond (CoA bond) which can be converted to a P bond by specific enzymes.

e.g. succinate thiokinase (another important enzyme we will learn about) succinyl CoA + GDP + Pi Succinate + GTP + CoA

or acetyl kinase acetyl CoA + Pi Acetyl P + CoA (+ADP) acetate + ATP + CoA. The name kinase suggests that the enzymes are involved in ATP synthesis. Most organisms are capable of carrying out these types of reactions and they are the major source of ATP in cells grown anaerobically in the dark but they do not contribute much ATP in cells that are grown aerobically of under photosynthetic conditions. III Oxidative phosphorylation ATP formed when protons are allowed into the cell through a membrane bound enzyme called F1Fo ATPase. (see Brock). F0 is a transmembrane protein and F1 is the ATPase enzyme that couples the energy from the proton translocation to ATP production. This uses pH of the PMF and is the major way of producing ATP for aerobically growing cells. IV Photophosporylation ATP formed from light reactions in bacteria and plants. The ATP is produced in the same way as for oxidative phosphorylation except the proton gradient is generated differently.