biochem quiz 2
Name the two primary electron carriers/donors for fuel oxidation. For each, give the name of both the oxidized and reduced forms of the carrier or donor.
(answer is three of them instead of two !) NAD+, FAD, and Acyl CoA are the fuel carriers/donors and they're in oxidized form. Reduced form is NADH + H, FADH2, and Acetyl CoA.
Indicate how ATP regulates the citric acid cycle (whether it inhibits or stimulates enzymes in the cycle). Indicate what step in the cycle it affects and the advantages this effect has for the cell's function(s)
- Generally, high ATP levels are going to inhibit progression of the CAC. When we have high ATP, we don't need any more. We would rather try to store the early intermediates as glycogen or use the intermediates of the CAC to make more cellular building blocks (amino acids, nucleotides, lipids, etc). - ATP inhibits pyruvate dehydrogenase (converts pyruvate to NADH + CO2 + acetyl-CoA) - ATP inhibits isocitrate dehydrogenase (converts isocitrate to alpha-ketoglutarate + NADH + CO2). When ATP levels are high, the cell doesn't need high energy electrons in the form of NADH to run the electron transport chain (to make more ATP). - Instead of reacting with isocitrate and transferring some of that energy to NADH, the cell can store all of that E in the form of citrate until it is needed later. - When the enzyme is inhibited, the reactant isocitrate builds up and will start being re-isomerized to citrate (no E lost). - Citrate can leave the mitochondria and inhibit an earlier step in glycolysis by inhibiting phosphofructokinase; this enzyme does the "committing step" of glycolysis, the conversion of fructose-6-phosphate to fructose-1,6-bisphosphate (PFK). - When E levels are high, isocitrate is converted back to citrate which is then transported out of the mitochondria where it can inhibit PFK and slow down glycolysis. - ATP also inhibits the next step in the TCA which is then catalyzed by alpha-ketoglutarate dehydrogenase (converts alpha-ketoglutarate to succinyl CoA).
Describe the ways in which oxaloacetic acid (OAA) can be utilized, in addition to its role in the citric acid cycle. Indicate how OAA can be "replenished" for the citric acid cycle.
- Oxaloacetate is normally reacting with acetyl-CoA to generate citrate at the beginning of the CAC. - Oxaloacetate can also be converted to phosphoenolpyruvate during the early steps of gluconeogenesis - Additionally, oxaloacetate can be utilized in the synthesis of aspartate from which other AA are made, in addition to certain purines and pyrmidines. - The # of oxaloacetate molecules can run low; if the cell runs low on ATP and needs to ramp up the CAC, oxaloacetate can be generated from pyruvate by the enzyme pyruvate carboxylase.
Describe all of the "competing interests" there are for pyruvate and explain, generally, why the regulatory coordination of these interests is important for the cell's various functions.
- Pyruvate can be utilized to make acetyl-CoA - Pyruvate can be converted to oxaloacetate for use in the CAC - Pyruvate can be used during gluconeogenesis (by first being converted to oxaloacetate) - Pyruvate is converted to acetylaldehyde then ethanol during alcoholic fermentation in microbes or to lactate during lactic acid fermentation, both with the goal of regenerating NAD+ during anaerobic conditions - Pyruvate is at the nexus of several energy-generating and energy-storing pathways. Regulation of pyruvate's fate is crucial for wasting as little energy as possible during metabolic flux.
Explain the overall function of the citric acid cycle and specify all the products it generates. Explain the importance and functions of each of these products.
- The CAC produces NADH, FADH2, and ATP/GTP by harnessing the energy released during breakdown of pyruvate to CO2 - Two carbons from pyruvate are transferred to Coenzyme A to create acetyl-CoA. Acetyl-CoA donates two carbons to the four carbon molecule oxaloacetate to create the six carbon citrate molecule (highest energy molecule in the CAC) - NADH and FADH2 carry E in the form of high E e- to the ETC, which are used to create the proton gradient across the inner mitochondrial membrane - ATP/GTP are used as general energy carriers to be utilized in all of the reactions in this class. - In some organisms, CAC generates ATP, in some GTP which is then converted to ATP at no E cost. - Two molecules of carbon dioxide are also released, which is how the two carbons are lost during the CAC - Oxaloacetate is regenerated after one run of the CAC, which can be used to make another citrate molecule when it receives two carbons from another acetyl-CoA
For each of the two stages of glycolysis, summarize briefly what happens in that stage.
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For the first part of the stage 1 of glycolysis (steps 1-3) name the two enzymes that are regulated and specify which reactions those two enzymes catalyze, i.e. the names of the substrates and products that are involved.
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For the second part of stage 1 of glycolysis (step 4), name the two products that are produced, and which enzyme is involved in their production. Also, indicate which of the two products is actually utilized in stage 2 of glycolysis and how the other product eventually can also enter the third stage of glycolysis; give name of the enzyme involved in this step (step 5).
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Give the name of the enzyme that catalyzes the final reaction in glycolysis, which reaction it catalyzes (substrates and products), including which high energy molecule is produced in the reaction.
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Name the two primary electron carriers/donors for fuel oxidation. For each, give the name of both the oxidized and reduced forms of the carrier or donor.
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Write out the net reaction of glycolysis, showing all the net inputs and outputs. In addition, indicate in which steps the ATPs and NADHs are produced.
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Name three disaccharidases and indicate what substrates they act on
1. Maltase: digests to 2 molecules of glucose 2. Sucrase: digests to glucose and fructose 3. Lactase: digests to glucose and galactose
Give the substrates and products for the first step in gluconeogenesis and indicate where in the cell this reaction takes place. Speculate as to why this reaction occurs in this location.
1st step: The substrates are pyruvate, CO2, ATP, and H2O and pyruvate carboxylase catalyzes this reaction into the products oxaloacetate, ADP, a phosphate, and 2 protons. The first step of Gluconeogenisis takes place in the mitochondria because it uses ATP.
Affinity Chromatography
A specific protein to be isolated based on its specific binding to a ligand attached to beads in the column Used for purifying a specific protein -Ligand will be attached to the bead and that ligand will have a high affinity for the specific protein Ex: glucose attached to the bead and the ligand will have a high affinity for the glucose -Glucose is soluble, so it can attach and unattached if needed. The bond is not permanent and that is more preferred
Explain what the following would indicate about the structure of a fatty acid: A. 18:0 B. 18:1 w-3 C. 18:2 cisDelta9 cisDelta12
A. 18:0 - 18 carbons no double bonds B. 18:1 w-3 - 18 carbons, double bond from omega 3 to omega 4 C. 18:2 cisdelta9 cisD12, 18 carbons, polyunsaturated cis
Describe the functions of each of the following enzymes in the digestion of large polysaccharides like glycogen and starch, indicating the specific bonds they cleave and the products of the reactions they catalyze. A. a-amylase B. a-glucosidase C. maltase D. dextrinase
A. A-amylase: cleaves alpha-1,4 bonds The first thing that starts breaking down large polysaccharides. -Generates alpha limit dextrin -Makes glucose molecules that are attached to others by branches. -Also creates smaller oligosaccharides (maltotriose or fragments of 4-6 glucose molecules) that are too small to get cleaved by a-amylase B. A-glucosidase: Cleaves alpha-1,4 bonds of maltotriose and other oligosaccharides to turn them into individual glucose C. Maltase: Cleaves alpha-1,4 bonds of maltose to separate the glucose molecules D. Dextrinase: Cleaves alpha-1,6 glycosidic bonds Cleaves those branches and makes maltose molecules (2 glucose molecules attached to each other)
Describe the role(s) of each of the following in the regulation of glycolysis and/or gluconeogenesis, indicating whether they stimulate or inhibit the pathway(s.) In addition, provide a rationale for each effect in terms of the advantages for the cell's functions (production of useful energy, storage of energy, biosynthesis of macromolecules, blood glucose regulation.) A. ATP B. ADP C. AMP D. Fructose 2,6 bisphosphate
A. ATP - ATP inhibits glycolysis and stimulates Gluconeogenisis (storage of energy). B. ADP - ADP inhibits Gluconeogenisis (production of useful energy). C. AMP - inhibit Gluconeogenisis and stimulate glycolysis (production of useful energy). D. Fructose 2,6 bisphosphate - Stimulates glycolysis and inhibits Gluconeogenisis (production of energy).
Indicate how each of the following affect the activity of the enzyme named in question #10 and provide a rationale for the effect in terms of overall cellular function (i.e. what advantages does this effect have for the cell?) A. ATP B. ADP C. NADH D. acetyl-CoA
A. ATP - inhibits pyruvate dehydrogenase by activating a kinase that phosphorylates PDH B. ADP - stimulates PDH C. NADH - inhibits PDH D. acetyl-CoA - inhibits PDH
Define the following with respect to a biochemical pathway and the conditions in which each is typically found: A. commitment step B. feedback inhibition C. feedback stimulation
A. commitment step - the first reaction that sets the biochemical pathway going B. feedback inhibition - the final product often negatively regulates enzyme for first step by binding and inhibiting its activity. if we have less of the final step, then there will be less of it to bind to the enzyme and inhibit it and the enzyme will be active. C. feedback stimulation - If both substrates needed to make final product, if low on one substrate F the other one I can stimulate F's allosteric enzyme to eventually make more of F so that I and F can make final product K. F can also stimulate I's allosteric enzyme.
For each of the disaccharides below, name the sugar monomers of which it is composed and the type of glycosidic linkage involved (which carbon atoms are involved and their orientation.) A. sucrose B. lactose C. maltose
A. sucrose: alpha Glucopyranosyl-beta-fructofuranose with a glycosidic linkage between alpha C1 and beta C2. B. lactose: beta-galactopyranosyl-alpha-glucopyranose with a glycosidic linkage between beta C1 and C4. C. maltose: alpha-glucopyranosyl and alpha-glucopyranose with a glycosidic linkage between alpha C1 and C4.
Indicate how ADP regulates the citric acid cycle (whether it inhibits or stimulates enzymes in the cycle). Indicate what step in the cycle it affects and the advantages this effect has for the cell's function(s)
ADP stimulates pyruvate dehydrogenase as well as isocitrate dehydrogenase. Both of these actions help push the reactions of the citric acid cycle forward at a faster rate when energy levels are low
Give the two major ways in which the primary product of pyruvate oxidation, Acetyl-CoA, can be utilized by the cell. Also, indicate a general mechanism, in addition to the oxidation of pyruvate, that can be utilized for making acetyl-CoA.
Acetyl CoA can be used by the cell to make lipids for storing energy and it is the major input of the citric acid cycle that carries the two carbons.
Name the 2 major kinds of fermentation. For each type, give the major end product(s) generated by the process.
Alcohol fermentation you end w ethanol and NAD+. and lactic acid fermentation end w lactate and NAD+. NAD+ allows glycolysis to continue.
Distinguish between sugars called aldoses and sugars called ketoses.
Aldoses are sugars that has a carbonyl attached to its terminal carbon. Ketoses have a carbonyl group attached to a carbon that is not the terminal carbon.
For affinity chromatography, describe the components of an affinity resin (beads)
Bead (matrix) non-reactive -A spacer will be between the ligand and bead, the ligand will bind to the specific protein with high specificity
Draw the basic structure of a fatty acid.
CH3- CH2- CH2- CH2- C=O=O
Name the two important satiety signals in mammals. For each, indicate in which tissues/organs they are produced, what tissues/organs they act on, and what their effects are, behaviorally and physiologically.
Cholecystokinin (CCK) Produced: By the intestine Acts on: The brain Effects: -Increased satiety in the brain -Decreased food intake -Decreased body weight Glucagon-like peptide (GLP-1): Produced: By the intestine Acts on: The pancreas, which acts on the brain (hypothalamus) Effects: -Increased satiety in the brain -Increased insulin secretion -Increased insulin biosynthesis -Increased beta-cell proliferation -Increased beta-cell survival
Distinguish between cis- and trans- unsaturation of fatty acids. Speculate as to why trans-fats are considered less healthy than cis-fats.
Cis unsaturation (most fatty acids in a biological system) has a bulky, kinked configuration making it less compact and therefore more fluid at room and body temperature. Trans unsaturation of fatty acids shows a linear, compact, and dense structure at room temperature. Usually show up for brief times in most biological systems. Trans-fats are considered less healthy because they can build up in our arteries and veins because of how compact they are, causing aggregates or solid masses that can increase risk of cardiovascular disease because it is harder to break them down at room and body temperature.
Name two irreversible enzyme inhibitors, indicate generally how they function and on which enzymes, and explain why their inhibitory effects on their target enzymes are irreversible.
DIPF is an irreversible enzyme inhibitor acting on the enzyme Acetylcholine Esterase by binding to it's serine's oxygen in the active site. DIPF is a nerve gas that blocks serine from being able to interact with the neurotransmitter acetylcholine therefore the nervous system is irreversibly damaged. Penicillin is an irreversible enzyme inhibitor of a bacterial enzyme transpeptidase. In a bacteria's cell wall, peptidoglycan is composed of sugars, tetrapeptide, and pentaglycine bridge. The bond between the tetrapeptide and the pentaglycine bridge is created by transpeptidase. In transpeptidase's active site, penicillin will form a covalent bond with the oxygen of the serine, blocking the active site. This makes the bacteria unable to build a cell wall.
Explain the purpose of ELISA and describe the similarities and differences between indirect ELISA and sandwich ELISA.
ELISA is a plate-based assay technique designed for detecting and quantifying soluble substances such as antibodies and hormones. Indirect ELISA -Used to detect the presence of antibody -The production of color indicates the amount of antibody to a specific antigen -The first antibody: Binds to the protein of interest -The second antibody: Binds to the first antibody and carries a label so it can be detected. - the well is coated with a protein sample (which may include protein of interest) Sandwich ELISA -Used to detect antigen rather than antibody -Production of color indicated the quantity of antigen -The protein of interest is directly bound by the capture and the detection antibody.
Indicate how energy charge is calculated and indicate the effects that high and low energy charge has, in general, on catabolic and anabolic reactions.
Energy charge regulates metabolism. At high levels of ATP, the energy charge is high the need for ATP decreases and it inhibits catabolic (exergonic releases ATP) or pathways that make ATP. It stimulates anabolic pathways (endergonic) or pathways that use ATP as it becomes more available.
Explain why "single molecule" studies of the enzyme can give a more complete understanding of the function of an enzyme, as compared to "ensemble" studies, in which a large population of enzyme molecules are studied together. What do the results of the "single molecule" studies reveal with respect to the structure of a particular protein?
Enzymes are only activated in very specific environments which is why it would be better to study them individually rather than as a whole. For example, if there is a slight change in pH or temperature the enzyme can denature.
For both prokaryotes and eukaryotes, name the cellular compartment in which each of the following occur: A. glycolysis B. gluconeogenesis C. pyruvate oxidation D. citric acid cycle E. electron transport chain/chemiosmotic synthesis of ATP
Eukaryotes do everything either external to the mitochondrion (glycolysis, gluconeogenisis) or inside the mitochondrion (pyruvate oxidation, citric acid cycle, electron transport chain/chemiosmotic synthesis of ATP). Prokaryotes do everything in the cytoplasm (glycolysis, citric acid cycle, gluconeogenisis) and on the plasma membrane (pyruvate oxidation, electron transport chain/chemiosmotic synthesis of ATP).
Describe the reactions involved for forming the most common cyclic form of glucose (a pyranose) and for forming the most common cyclic form of fructose (a furanose) from their respective open-chain (straight chain) forms. For each cyclic form, also indicate how many carbons are found in the ring and how they are numbered.
For alpha glucopyranose, the O on the OH group will form a covalent bond with the carbonyl C1 carbon. The cyclic form will have 5 carbons in the ring and the 6th carbon is above the ring and the new OH is below the ring plane. For alpha fructofuranose, the O on the OH group will form a covalent bond with the carbonyl C2 carbon. The cyclic form will have 4 carbons in the ring, the 1st and 6th C are above the ring and the new OH is below the ring plane.
Indicate the ways in which gluconeogenesis differs from the exact reverse of glycolysis.
From pyruvate to phosphoenolpyruvate there is a step that makes OAA with pyruvate carboxylase and then phosphoenolpyruvate with phosphoenolpyruvate carboxykinase. Also, from fructose 1,6 bisphosphate to fructose 6-phosphate the enzyme fructose 1,6-bisphosphatase is used. And then from glucose 6-phosphate to glucose the enzyme glucose 6-phosphatase is used instead of hexokinase.
For the second part of stage 1 of glycolysis (step 4), name the two products that are produced, and which enzyme is involved in their production. Also, indicate which of the two products is actually utilized in stage 2 of glycolysis and how the other product eventually can also enter the third stage of glycolysis; give name of the enzyme involved in this step (step 5).
Fructose 1,6 bisphosphate is cleaved into DHAP and GAP by aldolase. GAP is used in stage 2 and DHAP can eventually be turned into GAP by Triose phosphate isomerase which is the fifth step.
Explain why gluconeogensis is not simply the reverse of glycolysis from an energetic standpoint, and indicate what compensates for this difference.
Gluconeogenisis actually uses 6 NTP's as opposed to the reverse of glycolysis that only uses 2 ATP. Gluconeogenisis is more exergonic and the reverse of glycolysis is endergonic and doesn't produce enough energy to drive Gluconeogenisis.
Describe how monosaccharides are taken up into intestinal cells
Glucose, galactose, and fructose are transported to the wall of the small intestine and into the portal vein, which takes them into the liver From Jessica Law: -Once large polysaccharides + disaccharides are broken down into monosaccharides in the gut, special transporter proteins take up monosaccharides into intestinal cells -SGLT (Sodium-glucose transporter) transports glucose or galactose + a Na+ ion at the same time into the intestinal cell -Glut5 transports fructose into the intestinal cell -Afterward: Intestinal cell uses some monosaccharides (e.g. glucose) to make ATP for itself or store as glycogen -Transports excess (glucose + galactose + fructose) out of the cell & into the blood via Glut2
Name the enzyme, found in liver, that catalyzes the last reaction of gluconeogenesis, and indicate where in a liver cell this enzyme is located. Explain why the ability of liver cells to carry out this last step is important for distributing glucose to other parts of the body.
Glucose-6-phosphatase is the enzyme in the liver that catalyzes the last reaction of Gluconeogenisis - glucose-6-phosphate into glucose. Glucose 6-phosphatase is a membrane bound protein in the membrane of the ER in the liver cells. Glucose then can leave the liver cells and be sent out of the liver and into the blood. This enzyme can also convert glycogen to glucose.
For the first part of the stage 1 of glycolysis (steps 1-3) name the two enzymes that are regulated and specify which reactions those two enzymes catalyze, i.e. the names of the substrates and products that are involved.
Hexokinase and phosphofructokinase are regulated. The substrates are glucose plus ATP and hexokinase catalyzes is this reaction to form the products glucose six phosphate plus ATP plus a proton. The substrates are fructose 6 phosphate plus ATP and phosphofructokinase catalyzes the reaction to produce fructose 1,6 bisphosphate plus ADP plus a proton.
Explain how hexokinase activity is regulated and how the regulation of PFK directly affects the regulation of hexokinase.
Hexokinase is inhibited by its product glucose 6 phosphate. When fructose 6 phosphate gets converted back to glucose 6P when PFK gets inhibited by increased levels of PFK. So glucose levels build up in the cell, inhibiting hexokinase. Excess glucose 6P is converted to glycogen to store excess glucose.
Explain, from a free energy standpoint, why coupling a reaction with ATP hydrolysis allows reactions to proceed that would otherwise not proceed. Also, indicate what types of cell activities can utilize the energy released by ATP hydrolysis.
Hydrolysis of ATP is endergonic but when we take the energy from ATP hydrolysis and couple it with an enzyme, we can use the energy to make product from substrate and the ATP shifts the reactants toward the product side before it reaches equilibrium. We now have an exergonic reaction. The more available useful energy the higher the energy charge in the cell and high levels of ATP inhibit catabolic pathways and stimulate anabolic pathways. Motion, active transport, biosyntheses, and signal amplification utilize energy from ATP hydrolysis.
Explain why, in the absence of oxygen, pyruvate undergoes fermentation, i.e. explain why fermentation is necessary when oxygen isn't available.
In the absence of oxygen, many cells use fermentation to produce ATP by substrate-level phosphorylation. Pyruvate, the end product of glycolysis, serves as an electron acceptor for oxidizing NADH back to NAD+, which can then be reused in glycolysis. Fermentation is necessary when oxygen isn't available because it is the only way way of harvesting chemical energy without using oxygen or any electron transport chain (in other words without cellular respiration.)
Describe the structure of the large polysaccharides glycogen, linear starch (amylose) and branched starch (amylopectin), as well as cellulose. Also, indicate how glycogen and branched starch (amylopectin) differ from each other and how they both differ from cellulose.
Large polysaccharides: Glycogen- large branched homopolymer of glucose, made up of about 14 glucose molecules, has alpha 1,6 glycosidic linkages attaching branches and alpha 1,4 glycosidic linkages attaching chains. Branched starch (amylopectin)- has alpha 1,6 glycosidic linkages attaching branches and alpha 1,4 glycosidic linkages attaching chains Linear starch (amylose)- unbranched starch, long chains of alpha glucose Cellulose- straight, beta glucose chains with just beta 1,4 glycosidic linkages. Branched starch is different from glycogen because it has way less branches so less alpha 1,6 glycosidic bonds. branched starch is how plants store glucose. They both differ from cellulose because cellulose is made up of straight, beta glucose chains with just beta 1,4 glycosidic linkages and with parallel chains above and below held together by H bonds, not covalent branching like branched starch and glucose. We can cleave alpha 1,4 with an enzyme and alpha 1,6 with another enzyme but we cannot cleave beta 1,4 linkages so it just goes through (fiber).
Indicate where most of the remaining reactions of gluconeogenesis take place and how the product of the first reaction is transported to this location.
Most of the remaining reactions of Gluconeogenisis occur in the cytoplasm. OAA gets made into malate to get transported out of the mitochondria and into the cytoplasm. In the cytoplasm its converted back into oxaloacetate.
Compare the structures of myoglobin and hemoglobin and indicate how differences in their structures account for differences in their functions.
Myoglobin is a single globin polypeptide with one O2 binding heme group. Hemoglobin has four globin polypeptides (2 alpha helices and 2 beta pleated sheets) and each of these has an O2 binding heme group (4). In a heme, there is an iron that is surrounded with a tetrapyrrole molecule with 4 nitrogens that connect to the iron in the middle. When iron binds to O2, the nucleas goes from being underneath the tetrapyrrole plane to in-plane. This shifts the whole conformation of the heme group and induces a shift in the other heme groups into a relaxed form to increase the affinity for O2 binding (cooperativity). Myoglobin only has one heme therefore when it binds to O2 it is not affecting any other hemes to increase their affinity. That is why hemoglobin is more able than myoglobin to give up it's O2.
Indicate how NADH regulates the citric acid cycle (whether it inhibits or stimulates enzymes in the cycle). Indicate what step in the cycle it affects and the advantages this effect has for the cell's function(s)
NADH inhibits pyruvate dehydrogenase, isocitrate dehydrogenase, as well as alpha-ketoglutarate dehydrogenase. When NADH levels are high, we don't need to generate more NADH (isocitrate dehydrogenase & alpha-ketoglutarate dehydrogenase produce NADH)
Name the major protease found in the mammalian stomach and its inactive form. Also, describe how the inactive form is activated. Finally, describe the functions of hydrochloric acid (HCl) in the mammalian stomach.
Name : Pepsinogen (inactive) & pepsin (active) How the inactive form is activated: When the zymogen pepsinogen is cleaved, the active form of the major protease found in the human stomach is generated. The function of Hydrochloric Acid (HCI) 1. HCI converts pepsinogen into a partially active form 2. It interacts w/ pepsinogen and cleaves a portion of pepsinogen 3. It makes pepsin, which does the breakdown of food 4.HCl lowers pH and kills digestive microbes, while digesting that is with the molecules that we ingested
Write out the net reaction of glycolysis, showing all the net inputs and outputs. In addition, indicate in which steps the ATPs and NADHs are produced.
Net inputs: 1 glucose molecule, 2 electrons, 2 ADP, 2 NAD+ Net outputs: 2 pyruvate molecules, 2 ATP, 2 NADH, 2 protons, 2 H2O ATP is produced when 1,3 biphosphoglycerate is turned into 3 phosphoglycerate. And when phosphoenolpyruvate is turned into pyruvate. NADH (2) is produced when GAP is turned into 1,3 biphosphoglycerate.
Describe the main ways that the activity of phophofructokinase (PFK) is regulated in muscle and in liver.
PFK is inhibited by the high energy charge in the cell caused by increased ATP, so muscle is at rest. PFK is stimulated by AMP, which causes a low energy charge in the cell to make more ATP. In the liver, if glucose levels are high then fructose 6 phosphate gets converted into F-2,6-biphosphate which then activates PFK. Adding ATP lowers the activity of fructose 2,6 biphosphate which then inhibits PFK. This is a feedforward regulation.
Describe the major difference between a polyclonal antibody and a monoclonal antibody.
Polyclonal antibody: -Mix of antibodies -Bind the same protein at different epitopes Monoclonal antibody: -Copies of the same antibody -Binds a protein at one epitope
Gel filtration chromatography (size exclusion chromatography)
Proteins in a sample are separated into fractions based on differences in their size. Homogenate is going to go through a filter with carbohydrate polymer beads -Beads have pores of various sizes (slots for. the samples to fit in and not fall through the bottom) Smaller molecules will enter beads and larger molecules will not As it's put through a filter, the smaller molecules will stay in the aqueous bead holes and the larger molecules will get filtered out
Describe the purpose and basis of SDS/PAGE and the basic steps involved in the technique.
Purpose: Separating proteins by size, identifying particular proteins by size, or by staining to detect all different proteins Polyacrylamide is better for separating proteins b/c the size of the pores is more uniform and allows for more separation of diff. Sizes Basis: In SDS/PAGE, proteins migrate through the gel toward the positive electrode since the proteins are treated with SDS (sodium dodecyl sulfate), which coats them in negative charge. 1) The gel is poured into the wells and is allowed to solidify 2) The samples of interest are then pipetted into the well 3) Charge is applied and is spread evenly across the gel 3a) Proteins in the gel will move towards the positive electrode 3b) Before they are put into the well they are treated with detergent sodium dodecyl sulfate (negatively charged) -It denatures them and completely coats them with negative charges 4)They'll move towards the bottom of the gel but at different rates (the bottom of the well is also the positive electrode side, to which they will gravitate) -Smallest will move faster & reach the bottom/ larger will move slower & remain at the top -Everything goes through the pores since smaller ones can move them more quickly -Friction between larger proteins and smaller pores
Explain the purpose of Western blotting and describe all the steps involved in the technique.
Purpose: To determine whether a sample contains a specific protein of interest (OR they can put: to compare samples for their relative levels of a specific protein of interest) Steps: A. Run proteins in the sample in SDS/PAGE. (Separates proteins in gel-based on differences in their molecular mass.) B. Transfer proteins from gel to a membrane/nitrocellulose C. Probe blot with a primary antibody that specifically binds the protein of interest. D. Probe blot with a secondary antibody that specifically binds the primary antibody and carries a detectable label. E. Run procedure for detecting label (e.g. colorimetric enzymatic reaction) 3. Explain the important roles of the molecule sodium dodecyl sulfate (SDS) in the procedure? A. Coats proteins in negative charges so they run on gel (and move from the gel to blot in transfer); B. Denatures proteins (dissociation of non-covalent interactions) C. Lyses cells
Give the net reaction of pyruvate oxidation, i.e .all the initial substrates and the final products of the pathway (not the intermediates.) Also, name the enzyme that catalyzes the pyruvate oxidation reaction.
Pyruvate + CoA + NAD+ ----> acetyl CoA + CO2 + NADH + H+ (double everything). Pyruvate dehydrogenase catalyzes the pyruvate oxidation reaction.
Give the name of the enzyme that catalyzes the final reaction in glycolysis, which reaction it catalyzes (substrates and products), including which high energy molecule is produced in the reaction.
Pyruvate kinase phosphorylates ADP and a proton into ATP and this catalyzes the substrate phosphenolpyruvate to turn into pyruvate plus that ATP.
Name and describe the three major mechanisms by which reversible enzyme inhibitors act on enzymes. For each mechanism, indicate and explain the effect such an inhibitor has on Vmax for the enzyme as well as the effect on KM.
Reversible mechanisms: Competitive inhibition - bind to active site and prevent substrate from binding. Similar structure to actual substrate but don't get converted to product. Increase inhibitors lowers activity of enzymes, but eventually we can outcompete inhibitor if we add a lot of substrate and this will raise Km but no change to Vmax because we can eventually reach the same capacity of the system to make product. Uncompetitive inhibition - bind only to ES complex to inhibit activity. Certain population of enzymes removed because they are inhibitors with a substrate in the active site so adding substrate doesn't overcome this mechanism. The capacity to make product is lowered because removing enzymes so Vmax lowered and Km is also reduced because we now need less substrate concentration to make it to 1/2 Vmax. Noncompetitive inhibition: bind separate site from active site. Also same affect as uncompetitive with Vmax decreased but no effect on Km. At low substrate concentrations, uncompetitive inhibitors will have a higher effect on the reaction rate/velocity than noncompetitive inhibitors because they require a substrate to have an effect. (graph with red line higher than noncompetitive yellow line) Noncomp. bind to anything it's independent of substrate.
Distinguish between saturated, monounsaturated, and polyunsaturated fatty acids. Among these, which are the most and least fluid? Explain.
Saturated: Fatty acid that has all H's on the chain. least fluid because Monounsaturated: Fatty acid that has one double bond. Polyunsaturated: fatty acid that has 2+ double bonds.Most unsaturated so most fluid especially if all in cis. Fluidity is their state at particular temperatures, it is increase by short chain length, unsaturation, and cis arrangement of double bonds.
Describe the effects of the hormones cholecystokinin and secretin.
Secretin: -Induces the pancreas to secrete Na+ HCO3- (sodium bicarbonate) into the intestine. -Sodium bicarbonate acts as a buffer to pick up protons. By doing that it neutralizes the pH of the stuff that's coming in (gastric acid, food, enzymes, hydrochloric acid) Cholecystokinin (CCK): -The hormone induces the pancreas to release inactive digestive enzymes in the small intestine and induces gallbladder bile salts -Those inactive digestive enzymes will get active once they reach the small intestine -Bile salts help break down fat and large lipid aggregates into smaller droplets
Ion-exchange chromatography
Separates proteins based upon differences in their net charge The beads are going to be charged either negatively or positively charged (OPPOSITE) -If negative: a positively charged protein will bind to the bead while the negatively charged protein will be filtered out -If positive; a negatively charged protein will bind to the head and the positively charged one will be filtered out Good at narrowing proteins into a smaller sample size; won't find the exact protein you want with this method
For each of the two stages of glycolysis, summarize briefly what happens in that stage.
Stage one ATP is utilized it is the energy investing stage. Stage two is the ATP producing stage it is the energy harvesting stage.
Indicate how succinyl-CoA regulates the citric acid cycle (whether it inhibits or stimulates enzymes in the cycle). Indicate what step in the cycle it affects and the advantages this effect has for the cell's function(s)
Succinyl-CoA is produced by alpha-ketoglutarate dehydrogenase from alpha-ketoglutarate. Succinyl-CoA inhibits its own creator, the enzyme alpha-ketoglutarate dehydrogenase. High levels of succinyl-CoA communicates to alpha-ketoglutarate dehydrogenase that the E requirements of the cell are low.
Plot typical "temperature vs. activity" and "pH vs. activity" graphs for enzymes and explain why they have the shapes they do.
Temp vs activity-higher temp more kinetic energy higher enzyme activity, above some temp enzyme will denature pH vs activity-depends on where the proteolytic enzyme is but the peak of their activity is when they are in the location near their ideal pH and drops off-pepsin low pH in stomach-chymotrypsin high pH in small intestine-buffers regulate the pH changes
Give the two other names of the citric acid cycle
The TCA (tricarboxylic acid cycle) and the Krebs cycle
Explain how high levels of muscle activity, creating locally low oxygen levels, create potentially high levels of lactic acid in muscle and indicate how the body can deal with this excess lactic acid so that it doesn't build up to toxic levels and so that additional energy may be extracted from it.
The body deals with excess lactic acid by moving it from our muscles, into our blood and into our liver and then by turning it into pyruvate in the liver through Gluconeogenisis and then glucose to then go back into the muscles to do more glycolysis and make a little bit more ATP.
Name the three amino acids that form the catalytic triad for chymotrypsin and describe the specific roles each of these amino acids play in the catalysis of peptide bond hydrolysis by chymotrypsin.
The catalytic triad of amino acids in chymotrypsin is made of aspartate, histidine, and serine. Aspartate: Stabilizes histidine with H bond between it's O and histidine's H. It keeps it in the correct position with it's negative charge on the carboxyl group. Histidine and Serine: Histidine acts as a base catalyst and accepts a proton from serine and creates alkoxide ion on serine. Serine acts as a strong nucleophile to cleave the peptide's peptide bond. First, histidine gets protonated from serine's OH group. Then serine's O attacks the peptide's carbon, cleaving it's peptide bond. Then the nitrogen on one side of the peptide will deprotonate histidine and leave with the proton and histidine is neutral. Then histidine is reprotonated by water, the rest of the protein leaves, and histidine deprotonates back to serine.
Name the factors that regulate the activity of pyruvate kinase and the effects each regulator has on the enzyme's activity.
The insulin signalling pathway activates enzymes (phosphotases) that will remove the phosphate from pyruvate kinase making it more active. Glucagon signalling pathway will activate a kinase that will add a phosphate to pyruvate kinase making it less active. Fructose 1, 6 P stimulates pyruvate kinase and ATP inhibits.
Draw the basic structure of a triacylglycerol and indicate the major function of triacylglycerols in organisms.
The major function of triacylglycerols in organisms is that they are the storage form of fatty acids. Adipocytes have a large droplet of triacylglycerols in them. ( 2 fatty acids attached to a glycerol attached to phophate and alcohol)
Name the major phospholipid from which most membrane phospholipids are derived and draw its structure.
The major phospholipid from which most membrane phospholipids are derived is phosphotidate.
Describe the overall function of gluconeogenesis and summarize the overall result of stage one and the overall result of stage two of gluconeogenesis.
The overall function of gluconeogenisis is when there is a lot of ATP we can convert pyruvate back to glucose-6-phosphate and in the liver all the way to glucose. The overall result of stage one is glyceraldehyde-3-phosphate from pyruvate. Stage two result is glucose from GAP and DHAP.
Describe the basic concept underlying the technique of cell fractionation by differential centrifugation and the purpose of the technique.
The purpose of cell fractionation by centrifugation: 1) Separates different components of the cell into different fractions 2) Help isolate proteins based on which part of the cell they are associated with Due to the specific molecular weight component of the cell, they have to centrifuge longer at higher speeds to separate each part in the pellet Technique: Break open the cells in a buffer solution -1st time: Low speed for a short amount of time, the nuclear fraction is the pellet -2nd time: centrifuge supernatant, spin for higher speed and longer time and the pellet will be the mitochondrial fraction -3rd time: centrifuge supernatant again, for higher speed and much longer time and the pellet will the microsomal fraction (plasma membrane, Golgi, ER) Remaining: Cytosol, which are soluble proteins
Explain the purpose of immunoprecipitation and co-immunoprecipitation and describe the major steps involved in the techniques.
The purpose of co-immunoprecipitation is to identify the proteins that interact or form a complex with the protein of interest.ue called______________________________ an antibody is used to isolate a protein of interest along with proteins with which it interacts or forms a complex. The purpose of the technique is to identify the proteins that interact or form a complex with the protein of interest. The purpose for immunoprecipitation: used to identify AND purify the protein of interest (for protein complexes) The procedure for both is an antibody is used to isolate a protein of interest along with proteins with which it interacts or forms a complex. Steps: 1) Add beads, which have antibodies of interest, to the solution or mix of proteins 2) Protein of interests binds to the beads after mixing for a while 3) Then a centrifuge is used, beads at the bottom b/c they are heavier 4) Supernatant at the top, which is a mix of proteins that we do not want 4a) That is pipetted out of the solution and discarded 5) We might have other proteins we don't want to the bottom, so we repeat the process with washing Note: interaction between proteins & antibodies are non-covalent interaction, not strong -So if we add denaturant, it will break the bonds -Antibodies and proteins are separated, when put in the centrifuge again beads will be at the bottom and supernatant at the top (which has the proteins of interest) Extra Step: can use a different method to get protein of interest, like western blot (if you have an idea for what the other proteins, besides your protein, are) or mass spectrometry (if you have no idea, you can use this to find each one) Imperfection: -False Negative: If protein of interest doesn't get detected even though its there -False Positive: if our protein interacts with another protein that it shouldn't interact w/by chance Major Difference: Antibody will pull down the protein of interest but it will have other other proteins bound to it too
Describe what characterizes the group of lipids called steroids.
They have a steroid nucleus made from three 6C rings with a 5C ring attached.
Describe how triacylglycerols are digested, in what form they are absorbed into intestinal cells and how they are then "packaged" for transport to the rest of the body.
Triacylglycerols are transported out of the intestinal cell & into the body via the lymphatic system (lymph nodes) primarily in molecular complexes called chylomicrons Digestion of triacylglycerol by the enzyme lipase produces 2 free fatty acids and monoacylglycerol in the lumen of the small intestine -Cleaves ester linkages of fatty acids using water (hydrolysis RXN) -Free fatty acids (w/ charged group on end) + monoacylglycerol molecules tend to form little droplets together w/ charged/polar groups outside & hydrophobic groups inside (micelles) -Micelles = taken from the lumen of the gut into the intestinal cell through FABP (fatty acid binding protein) -Transported by FATP (fatty acid transport protein) into the smooth endoplasmic reticulum (SER) -Fatty acids + monoacylglycerols reform into triacylglycerols & are packaged with phospholipids, cholesterol, and proteins into chylomicrons (TAG)
Draw a graph showing the dual effects of CO2 on hemoglobin function and describe and explain these effects. In addition, describe these effects and indicate how these effects are adaptive for organisms.
When CO2 diffuses across the body tissue into the the blood capillary and into a red blood cell, this addition of CO2 in the cell plus water pushes the equilibrium to the right to form carbonic acid H2CO3 and then this increasing amount makes them dissociate into a bicarbonate ion and a proton. When there is an increase in protons in the red blood cell, there is a drop in pH around this cell. Cells that are active in metabolism, making lots of ATP, generating CO2, they need an extra burst of O2 so hemoglobin has this mechanism where when the pH drops, it gives up more O2 (decreasing affinity).
Describe how the concept of reaction equilibrium is important for driving the efficient elimination of the waste product CO2 in the lungs.
When CO2 starts to decrease in the red blood cell, the equilibrium is pushed back to the left and the H2CO3 carbonic acid will turn into water and CO2 which will again be released from the cell.
Describe the mechanism by which 2,3 BPG affects hemoglobin function. Also, draw a graph showing the results of an experiment to test the effects of 2,3-BPG on oxygen saturation of hemoglobin.
When glycolysis is active, it is an indicator that the cell needs to make ATP so it needs a lot of O2. 2,3 BPG is a byproduct of glycolysis and when hemoglobin is in the presence of 2,3 BPG, it acts as an allosteric regulator of hemoglobin's oxygen affinity (allows it to give up more O2 at low concentrations). 2,3 BPG molecule has lots of negative charges and so it is found in the central cavity of hemoglobin where it has electrostatic interactions with the basic, positive, amino acids such as histidine and lysine. This will lower the affinity for O2 because it shifts hemoglobin to a more tense state and hemoglobin will give up more O2 for use in glycolysis.
For each of the following enzymes, give the name of its zymogen and describe how it is activated. A. Trypsin B. Chymotrypsin C. Carboxypeptidase D. Lipase
Zymogen Name A. Trypsinogen is the zymogen of trypsin, an enzyme that aids in the digestion of proteins B. Chymotrypsin (Chymotrypsinogen) C. Carboxypeptidase (pro-carboxypeptidase) D. Lipase (Pro-lipase) Activated by: A. Trypsin is activated by Enteropeptidase Trypsin Activates: B. Chymotrypsin (Chymotrypsinogen) -The enzyme trypsin cleaves the zymogen chymotrypsinogen to produce active chymotrypsin. C. Carboxypeptidase (pro-carboxypeptidase) D. Lipase (Pro-lipase)
Define the terms metabolism, catabolism, and anabolism
metabolism: Sum total of all chemical reactions in an organism. catabolism: Break down complex molecules into simpler molecules, with the release of energy anabolism: Reactions that build complex molecules from simpler ones (and typically need an input of energy)