Exam #2 Review

Exam #2 will cover all the material that has been presented in class since Exam #1 and up through the metabolism introduction. This includes eukaryotic cell structure / function, transport, the closed system growth curve, enzymes and the introduction to metabolism. As always, it is best to begin by studying your notes and then after you feel your study is complete, take some time to look through this review.

I. Eukaryotic cell structure / function A. There is a great deal of variance among eukaryotic cells - from protozoan cells to yeast cells to human cells. Fungi and (classically split into algae and ) are eukaryotic representatives of the microbial world. B. Structure of the eukaryotic cell. 1. Cytoskeleton - provides structure and shape of cell, three components: a. Microtubules - largest element of cytoskeleton, composed of hollow cylinders of tubulin, form mitotic spindles, cilia and flagella and cell “highways”. b. Microfilaments - smallest element of cytoskeleton, composed of actin, involved in motion (pseudopod formation). c. Intermediate filaments - very stable structural element, play a supportive role, composed of proteins including keratin. Practice: Microfilaments a. are a component of the cytoskeleton. b. are long, twisted polymers of a protein called actin. c. form eukaryotic flagella. d. are made of tubulin. e. a and b f. c and d 2. Nucleus a. Bound by both an inner and outer membrane. The space between the two membranes is called the perinuclear space. The membrane has large nuclear pores through which proteins can pass (Why is this important?) b. Linear pieces of DNA are packaged by wrapping one and three quarters times around a histone octamer to form a core particle. Neighboring core particles are linked by DNA. This linker DNA associates with another histone called H1. Together, the core particle, H1 and some of the linker DNA form a nucleosome. Histones are rich in what type of amino acid? How does the charge of DNA / histones affect this packaging process? Chromatin is the complex of DNA and proteins that together form the chromosomes. How does euchromatin differ from heterochromatin? c. A region within the nucleus, called the nucleolus, is the site of ribosome assembly. Practice: The nucleus a. is a double membrane sac containing DNA as is found in . b. Is a single phospholipid membrane sac containing prokaryotic DNA. c. is a smaller structure contained within the eukaryotic nucleolus. d. cannot transport molecules to the due to the double membrane barrier. 3. The Endoplasmic Reticulum a. Rough ER - Site of synthesis of proteins targeted for the membrane, for secretion or for specific organelles. (Studded with 80S ribosomes (*60S and 40S subunits)) What is the medical importance of the fact that prokaryotes have 70S ribosomes and eukaryotes 80S? b. Smooth ER - Site of lipid and steroid hormone synthesis, location of calcium ion storage. (No ribosomes cover the surface, thus smooth). *vesicles bud off of both of these organelles carrying the recently synthesized compounds. These vesicles are targeted for the Golgi. Practice: Which one of the following would be the site of synthesis and folding, but not final modification of a protein needed outside the cell? a. The Golgi apparatus b. The mitochondria c. The smooth ER d. The rough ER 4. The Golgi apparatus - molecules from ER are further modified (e.g. addition of carbohydrate or phosphate groups). Vesicles bud off of Golgi and carry modified molecules to their destination. The Golgi has a cis and a trans face that differ substantially from one another. Practice: The Golgi Apparatus a. is the site of modification of molecules from the ER. b. has an acidic pH and is filled with hydrolytic, digestive enzymes. c. is found exclusively in and algae. d. is considered the “powerhouse” of the eukaryotic cell. e. both b and c 5. Lysosomes - acidic interior, filled with digestive enzymes to hydrolyze macromolecules. 6. Mitochondria - powerhouses of the cell! Practice - draw this organelle and label all parts. Where are the components of the electron transport chain? Practice - Which one of the following organelles contains its own DNA genome and 70S ribosomes? a. lysosomes b. the endoplasmic reticulum c. mitochondria Which statement/s regarding mitochondria is/are FALSE?

a. Mitochondria are capable of converting CO2 into organic compounds. b. Mitochondria reproduce by binary fission. c. Nearly all eukaryotic cells contain mitochondria. d. Mitochondria contain their own DNA genome 7. Chloroplasts Practice - draw this organelle and label all parts. Where are the components of the electron transport chain? Practice - Both chloroplasts and mitochondria synthesize ATP using the electron transport chain, in what ways are these two organelles different (in terms of the origin of the electrons that fuel the electron transport chain)? Practice: Which of the following explains the origin of mitochondria and chloroplasts? a. The Chemiosmotic Hypothesis b. The Endosymbiont Theory c. The Fluid Mosaic Model d. Spontaneous generation 8. Plasma membrane In many ways the plasma membrane of a eukaryotic cell is similar to that of a prokaryotic cell (e.g Both are composed of a semipermeable phospholipid bilayer (fluid-mosaic). Both contain transport proteins. Practice - List the ways in which the plasma membrane of a eukaryotic cell is different than that of a prokaryotic cell. a. Endocytosis 1. - Used to engulf large particles - cell sends out long extensions called pseudopods. These surround debris and form a phagosome which fuses with a lysosome to form a phagolysosome (often termed cell eating). 2. pinocytosis a.) Macropinocytosis b.) Clathrin-dependent (Receptor mediated) c.) caveolae-forming Practice: Which statement/s is/are TRUE? a. Phagocytosis and pinocytosis are both types of endocytosis. b. Receptor mediated (clathrin-dependent) endocytosis is a type of pinocytosis. c. During phagocytosis, a phagolysosome is formed. d. During caveolae-forming endocytosis, no lysosome fusion occurs, thus many pathogens enter cells this way. e. all of the above *Remember that many pathogens exploit endocytosis to enter a host cell. b. Exocytosis 9. Flagella and cilia Practice: How are flagella different in prokaryotes and eukaryotes? All of the following are true of eukaryotic flagella EXCEPT a. eukaryotic flagella are composed of 9 pairs of microtubule doublets. b. eukaryotic flagella sometimes have lateral hairs called flimmer filaments. c. eukaryotic flagella are composed of a basal body, a hook and a filament. d. eukaryotic flagella function in motility by wave-like motion (base to the tip or tip to the base). Cilia a. are also called pili. b. are also called fimbriae. c. are shorter than flagella. d. move in two phases. e. a and b f. c and d *NOTE: It is a good test of understanding to compare features of prokaryotic and eukaryotic cells. Practice: Which statement does NOT correctly summarize a difference between prokaryotic and eukaryotic cells? a. The electron transport chain of aerobically respiring prokaryotic and eukaryotic cells is found across the cytoplasmic membrane. b. Whereas prokaryotes generally have a single chromosome, eukaryotes usually have more than 1 chromosome for the storage of genetic material. c. eukaryotic cells have a membrane-bound nucleus whereas prokaryotic cells have only a gel-like mass called the nucleoid. d. Whereas prokaryotic cells secrete enzymes to digest macromolecules, eukaryotes generally bring in large molecules via endocytosis. Which of the following is useful in distinguishing between prokaryotic and eukaryotic cells? a. the presence / absence of peptidoglycan b. the type of ribosomes used for the synthesis of proteins c. the presence of membrane-delimited organelles within the cytoplasm d. all of the above For the following 5 cell components, please determine whether they are generally present: a. only in prokaryotes b. only in eukaryotes c. only in bacteria d. in both prokaryotes and eukaryotes ____16) Endoplasmic reticulum ____17) A single circular chromosome ____18) N-acetylmuramic acid (NAM) ____19) diaminopimelic acid ____20) flagella

V. Microbial nutrition and growth A. Uptake of nutrients (transport) The diagram below summarizes the types of transport covered. Use this to review the differences / similarities of each type of transport.

Practice: Transporters using this type of transport require no energy but become saturated at high substrate concentrations. a. active transport b. simple diffusion c. facilitated diffusion d. none of the above *During what type of transport is a molecule modified as it is transported? B. Protein secretion - be familiar with the Sec-dependent pathway! C. Bacterial cells reproduce via binary fission: a parent cell replicates its DNA, elongates, a septum forms and eventually cleaves off two distinct daughter cells. With this type of growth, the increase in cell numbers is exponential. Practice: If a single cell lands on an agar plate and begins to divide, how many cells will there be after the tenth cycle of division? D. A researcher may also be interested in calculating the number of cells in a population of cells that originated from more than a single cell. The following equation can be used for this purpose and will be provided on the exam: n Nt = No X 2 where Nt = the # of cells in a population after a given time No = The original number of cells in a population n = the number of cycles of division (depends on doubling time) Practice: 100 cells of Vibrio fischeri are used to inoculate a large bottle of sterile media. If this bacteria has a doubling time of 2.5 h, how many cells are present in the culture after an overnight incubation (16 h)? C. In the lab, are generally grown in closed (batch) systems (Why are these systems called closed systems??). The Growth Curve describes growth in closed systems. Please spend some time reviewing this growth curve in your notes. Practice: During which growth phase do cells begin to synthesize secondary metabolites in response to increasing cell population and buildup of waste products? a. Lag phase b. Initial log phase c. Late log phase d. Stationary phase e. Death phase Practice: (T or F) All of the cells in a culture die during the death phase. Practice: Which statement/s regarding the stationary phase of growth is / are FALSE? a. It is during the stationary phase that culture growth stops. b. During the stationary phase, cells no longer synthesize any metabolites. c. The total number of viable cells stays constant during the stationary phase. d. Secondary metabolites are produced during the stationary phase. e. it is during the stationary phase that cells are most sensitive to antibiotics. f. b and e D. Why is growth in nature different from growth in a closed (batch) system?

Nutritional Factors that Influence Microbial Growth (What do microbes need in order to grow?) *Revisit your crossword puzzle! Not covered extensively on the exam but very important general knowledge to support your further learning. I will assume knowledge of terms like chemoorganohetertroph, chemolithoatutroph, psychrophile, acidophile etc.....

II. Enzymes A. Catalyze reactions by lowering the reaction activation energy. Enzymes lower the reaction activation energy by 1. holding the reactants in such a way that product formation is more likely. 2. stabilizing the transition state (a transition state is a high-energy complex that has a structure is somewhere between that of products and reactants). Practice: The following reaction curves both represent the same reaction, which curve depicts the enzyme catalyzed reaction?

Enzymes have an active site at which they bind specifically to their substrate. Enzymes either fit their substrate precisely (lock-and-key) or they change their conformation slightly to fit the substrate = induced fit. After the reaction has been catalyzed and the products are released, the enzyme remains unchanged. Sometimes, two enzymes may recognize the same substrate. What does this often lead to in a pathway? B. Allosteric regulation - Some enzymes have another site, in addition to the active site, called the allosteric site. An effector molecule binds to this site, changes the shape of the enzyme and in doing so, either inhibits or activates the enzyme. End products of many catabolic and anabolic pathways serve as allosteric regulators for enzymes in the pathways that produce them = feedback inhibition. C. Some enzymes are regulated when a chemical group is covalently bound to them. These enzymes are called interconvertible enzymes. In the example in the notes, this interconvertible enzyme is active when it has a phosphoryl group bound to it and inactive when it does not. The enzymes that covalently modify interconvertible enzymes are called converter enzymes. In the example in the notes, one converter enzyme acts to attach a phosphoryl group (thus activating the interconvertible enzyme) and another acts to remove that group (thus inactivating the interconvertible enzyme). Pyruvate kinase (remember from step 10 of glycolysis) is an enzyme that is extensively regulated by covalent modification. C. Some enzymes can only function with the assistance of a cofactor. An enzyme without its cofactor is termed an apoenzyme. When the enzyme has its cofactor it is termed a holoenzyme. Apoenzymes are not active. Cofactors are always non-protein components and can be split into two groups: essential ions and coenzymes. Coenzymes can be further divided into cosubstrates and prosthetic groups. Whereas cosubstrates can dissociate from the enzyme (like NADH - that can dissociate and carry it’s electrons to the ETC), prosthetic groups remain bound to the enzyme (like

FADH2 that can’t ever leave succinate dehydrogenase). Practice: Coenzymes are derivatives of a. minerals b. proteins c. lipids d. vitamins Practice: For her undergraduate research project a student is studying a very active enzyme. The student notices that when she treats the enzyme with a solution that removes metal ions, the enzyme suddenly becomes inactive. Explain to the student why this happens. D. Enzymes can be inhibited either competitively or noncompetitively. In competitive inhibition the inhibitor actually competes physically with the substrate for access to the active site. Practice: Which of the following is/are competitive enzyme inhibitors? a. sulfa drugs b. Magnesium c. NAD+ d. both a and b e. both b and c In noncompetitive inhibition, the inhibitor and substrate act at different sites. Competitive and noncompetitive inhibitors can inhibit either reversibly or irreversibly. Irreversible inhibitors bind covalently and effectively “kill” the enzyme. Penicillin is an example of an irreversible inhibitor. Reversible inhibitors bind to an enzyme in such a way that the enzyme is unaltered.

III. Metabolism - overview A. Catabolism = Harvesting energy released when a high-energy food molecule is BROKEN DOWN (oxidized, degraded). Glycolysis and the TCA cycle are basically catabolic pathways. However, because many of the intermediates formed in these pathways can serve as precursor metabolites, this gives these pathways a dual nature and we thus often call them amphibolic pathways. B. Anabolism = Energy (ATP) and reducing power (NADPH) are utilized to BUILD (synthesize) complex molecules from simple building blocks (e.g. the synthesis of amino acids). Practice: Biosynthetic reactions that require energy for the conversion of molecular subunits into larger molecules are called a. kinetic energy b. catabolic reactions c. precursor molecules d. anabolic reactions Practice: Concerning catabolism and anabolism a. they refer to reactions solely dealing with the synthesis of lipids. b. the intermediates of one serve as the reactants in the other. c. the energy gathered in one is utilized in the other. d. they refer solely to the reactions involved in the synthesis of proteins. e. b and c B. As can be seen by the definitions of catabolism and anabolism, ENERGY is a central theme in metabolism. The processes are all about gaining and utilizing energy. 1. Energy is never created or destroyed, it is simply converted from one form to another = The First Law of Thermodynamics. 2. Because reactions are constantly taking place within a cell, the total internal energy of that cell is constantly changing. The changes observe the First Law of Thermodynamics in that if the cell looses energy, the surroundings gains it and vise versa. The change in the total internal energy of a cell is seen in the form of a heat transfer which we represent as the change in enthalpy or ΔH. Is it more favorable for a system (cell) to gain or loose heat? 3. In addition to heat transferred when a reaction takes place, the nature of that reaction is also determined by The Second Law of Thermodynamics which basically says that NATURE SEEKS . The entropy (S) of the universe is always increasing. Is a negative or positive ΔS more favorable? 4. Thus, if a reaction is both releasing heat (ΔH is negative) and undergoing an increase in disorder (ΔS is positive), the reaction is spontaneous and exergonic (ΔG (free energy) is negative). A reaction may be spontaneous and exergonic if there is a decrease in disorder but the heat released must be great enough to make ΔG negative. In the same way, a reaction may be spontaneous and exergonic if heat is consumed as long as the increase in disorder still allows ΔG to be negative. ΔG = ΔH - TΔS 5. If ΔG is positive, the reaction is not spontaneous and is endergonic. Practice: Exergonic reactions a. occur when the value of ΔG is negative. b. are spontaneous c. occur when the value of ΔG is positive. d. both a and b e. both b and c C. The First Law and the relationship between photoautotrophs and chemoheterotrophs. The relationship between photoautotrophs and chemoheterotrophs is all about energy conversions and the First Law of Thermodynamics. Energy is conserved when kinetic light energy is converted into potential chemical energy (in the form of high energy bonds of organic “food” compounds) by photoautotrophs. Energy is again conserved when the high energy bonds of these “food” molecules are broken and converted to chemical energy powering the cellular functions of a chemoheterotroph. D. Metabolic pathways are comprised of a series of reactions. These pathways must be dynamic and coordinated so that cells can respond to changes in environment. Each reaction is catalyzed by a specific enzyme. Every enzyme-catalyzed reaction represents a potential point of regulation (inhibition or activation). In catabolic pathways the starting compound (an energy source (food)) is continually oxidized and the electrons are gathered by electron carrier molecules. The reduced electron carriers represent ENERGY, as they can drop off their electrons at the electron transport chain and they can be used to create the proton motive force and ATP (= oxidative phosphorylation). The energy released from exergonic reactions during catabolic pathways (e.g. breaking the high energy potential bonds in a food molecule during glycolysis) can be used to create metabolic intermediates with high energy, enough to phosphorylate ADP and create ATP = substrate-level phosphorylation. Practice: The name given to the reaction involving removal of electrons or hydrogen atoms from a compound is termed a. glycolysis b. reduction c. oxidation d. metabolism

[Note - decarboxylation means loss of CO2.] Some questions may ask you to be able to make a differentiation between substrate-level and oxidative phosphorylation. Remember that the phosphorylation of ADP to make ATP requires energy. If the energy comes from a high-E molecule (e.g. 1,3-BPG) than this is called substrate-level phosphorylation. If,

instead, oxidation of NADH / FADH2 forms the PMF and the PMF provides the energy to make ATP, this is called oxidative phosphorylation (OX PHOS). E. Electron carriers - When reduced, NAD+, FAD and NADP+ all carry 2 electrons. NADH and FADH2 carry their electrons to the electron transport chain to be used in ATP production. NADPH uses its electrons in anabolic (biosynthetic) reactions. Practice: In lecture, we talked about metabolism as we ate donuts. The glucose molecules in these donuts were gradually oxidized by catabolic pathways. As the glucose molecules were broken down by oxidation reactions, electron carrying molecules such as ______were reduced. These electron carrying molecules can then deliver their electrons to the electron transport chain. a. NAD+ and FAD b. ADP and AMP

c. CO2 d. hexokinase F. ATP (adenosine triphosphate) - UNIVERSAL ENERGY CURRENCY OF THE CELL! Practice: The phosphoanhydride bonds of ATP are high in energy because a. of charge / charge repulsions between the negatively charged oxygen atoms of the phosphoanhydride groups. b. the products of hydrolysis are better solvated than ATP itself. c. the products of hydrolysis are more stable than ATP itself. d. all of the above It is not necessary to memorize the structure of ATP, however it is important to note that ATP has a great deal of potential energy stored in its high energy phosphoester and phosphoanhydride bonds. When one of these high energy bonds is broken, a great deal of energy is released (exergonic reaction-, ΔG is negative). This energy can be used to power endergonic reactions (e.g. the phosphorylation of glucose in step 1 of glycolysis). When ADP is converted to ATP by phosphorylation, energy must be invested (endergonic reaction, ΔG is positive). Understanding the table of Standard Free Energies of Hydrolysis: 1. The reaction that is third from the bottom in the table is the hydrolysis of ATP. Looking at the free energy of hydrolysis of ATP to form ADP, we see that it is an exergonic process that releases 30 kJ/mol (ΔGº’ = -30 kJ/mol). This means that the opposite reaction in which ATP is phosphorylated is endergonic and requires the input of 30 kJ/mol (ΔGº’ = 30 kJ/mol). 2. Notice that there are many metabolites found above ATP in the table. For example, the metabolite called phosphoenolpyruvate (which you’ll recognize as an intermediate in glycolysis), has a free energy of hydrolysis of -62 kJ/mol. This means that Phosphoenolpyruvate could very easily and exergonically transfer a phosphate group to ADP to for ATP. In fact there would be 32 kJ/mol left over (-62+30 = -32 kJ/mol). This is in fact what happens in step 10 of glycolysis! 3. Also notice that there are some metabolites below ATP on the table, ATP could easily transfer a phosphoryl group to these metabolites. For example, ATP could transfer a phosphoryl group to glucose forming glucose 6-phosphate (in fact, this is the first step of glycolysis - HOW MUCH ENERGY IS RELEASED WHEN THIS NET REACTION HAPPENS?). D. During catabolism, many intermediates are formed. These intermediates can sometimes be syphoned off and used as building blocks in anabolism. Intermediates that can be used this way are termed precursor metabolites.