CHEM 301/ CHEM301 Final Examination | Biochemistry – 2026/2027 Academic Year – Questions with Verified Answers and Elaborated Solutions

CHEM 301/ CHEM301 Final Examination | Biochemistry – 2026/2027 Academic Year – Questions with Verified Answers and Elaborated Solutions Q: Which ONE of the following pairs of functional groups can interact by forming a hydrogen bond at physiological pH? Answer - Two amides. - Two phenyl rings. - Two aldehydes. - Two esters. Q: What type of functional group links amino acids together to form a protein? Answer - thioester bond - hydrogen bond - glycosidic bond - amide bond Q: What role does the hydrophobic effect play in protein folding? Answer - It allows for proteins to fold when some amino acids avoid contact with water. - Proteins only fold when they become insoluble in water. - The second law of thermodynamics provides energy for protein to fold. - It causes an overall decrease in the entropy of water. Q: Proteins that catalyze chemical reactions are classified as: Answer - proteases - enzymes - hormones - catalases Q: Hormones can belong to which ONE of the following categories? Answer - Lipids - Nucleic Acids - Carbohydrates - Vitamins Q: What is the take home message for BCH210H? Answer - The structure of a molecule is essential for its function. - All proteins require vitamins for their function and are essential for human health. - Biochemistry is all about the chemical reactions in the cell. - Cells rely primarily on proteins for structural support. Q: Which ONE of the following biomolecules has the most complex structure? Answer Answer - An amino acid - A polysaccharide - A molecule of water - A nucleotide Q: Which ONE of the following non-covalent forces can contribute the MOST to a molecule (eg. drug or cofactor) that interacts with a soluble protein? - A disulfide bond - Van der Waals forces - The hydrophobic effect - Ionic interactions Q: In biochemistry, PDB stands for: Answer - Protein Data Bank - Platform to Deduce Binding partners - Polypeptide Data Base - Program for Documenting Biomolecules Q: Which ONE of the following pairs of amino acids (represented by 3 letter codes) have side chains that can form a salt bridge? Answer - Cys - Cys - Lys - Asn - Arg - Ser - His - Glu Q: Which ONE of the following tri-peptides (amino acids represented by their 1-letter code) would have a titration curve with 4 buffering regions? Answer - F-A-R - S-A-D - P-E-P - M-I-N Q: Which ONE of the following statements is CORRECT regarding amino acids? Answer - They can form covalent and non-covalent interactions. - They are considered essential if they are found in every protein. - They join together to form polypeptides via amine bonds. - They only contain carbon, hydrogen, oxygen and nitrogen. Q: What is the approximate isoelectric point (pI) of Cysteine (pKas = 1.7, 8.3, 10.8)? Answer - 5.0 - 6.9 - 9.6 - 8.3 Q: How many of the 20 standard amino acids are zwitterions at physiological pH=7.4? Answer - 11 - 16 - 15 - 5 Q: Which ONE of the following polypeptides only contains letters that represent the one- letter codes for the 20 standard amino acids? Answer - J-U-N-E - F-E-B-R-U-A-R-Y - D-E-C-E-M-B-E-R - A-P-R-I-L Q: What is the correct 3 letter code for Glutamine? Answer - Gln - Glq - Gla - Glu Q: Which ONE of the following groups of amino acids (represented by their 3-letter codes) is least likely to be found on the surface of a soluble protein? Answer - Ala, Glu, Ser - Asp, His, Gln - Cys, Leu, Phe - Asn, Lys, Thr Which ONE of the following statements is CORRECT regarding chirality and amino acids? Answer - Chirality arises due to the presence of 4 different carbons in the structure. - The 20 amino acids each contain a chiral alpha carbon. - Amino acids are chiral because they have different side chains. - A chiral amino acid will have an L and D isomer. While purifying your protein of interest from a crude extract, you forget to label the collection tubes and mix up which samples contain the void volume and which ones eluted from the column. Which ONE of the following techniques is the best choice to determine which fraction you should use for subsequent experiments? Answer - UV spectroscopy - Dialysis - Bradford assay - Western blotting Which ONE of the following is CORRECT regarding the differences between an alpha helix and beta strand/sheet? Answer - They can be found in different motifs contributing to the level of tertiary structure. - Hydrogen bonds can form between amides in an alpha helix but not between amides in a beta sheet. - Amino acids can only exist in alpha helices or beta strands based on their properties. - A stretch of 20 amino acids would have a more extended structure in an alpha helix. Which ONE of the following types of secondary structure will the following polypeptide (amino acids represented by their 1-letter code) most likely form? W-A-V-T-F-K-I-G-Y-C - beta strand - beta turn - Amphipathic alpha helix - random coil Which ONE of the following statements is CORRECT regarding the amino acids found in a protein? - The composition of amino acids dictates the secondary structure that will form. - A protein's amino acid sequence doesn't necessarily tell you its function. - All proteins include the 20 basic amino acids in their primary structure. - Proteins in the same family of proteins have identical amino acid sequences. You are wish to study the structure of a brand new protein of unknown structure and function, but are having trouble purifying it from its endogenous source. Which ONE of the following would be useful to assist in its purification? - Co-immunoprecipitation with another protein. - The inclusion of a GST tag at the N-terminus. - Dialysis to change the pH of the buffer to the pI. - Treating the crude lysate with formaldehyde. Which ONE of the following statements is CORRECT regarding the role of hydrogen bonds for protein structure? - These covalent interactions are necessary for the formation of tertiary structure. - Hydrogen bonds between side chains allow for alpha helices to form. - Amino acids are joined by hydrogen bonds to form a polypeptide. - They can contribute to the formation of quaternary structure. You are monitoring the elution of your protein of interest from a size exclusion column and detect an absorbance of 0.45 at 415 nm in a 1 cm cuvette. Given a molar absorptivity of 15,000 L mol-1 cm-1 , what is the concentration of protein present in your fraction? - 30 uM - 6.75 uM - 67.5 nM - 3.0 mM Which ONE of the following statements is CORRECT regarding the methods used to determine a protein's sequence? - The chemical cleavage in Edman degradation limits how many amino acids can be sequenced. - Edman degradation is an effective way of detecting post-translational modifications. - Mass spec can only deduce the molecular weight of a polypeptide chain, not its identity. - Mass spec can only be done after larger chains are cleaved into smaller fragments. Protein X has a pI of 9.0 and is in a buffer with a pH = 7.4, along with a mixture of other proteins. You decide to try and purify it using a DEAE (diethylaminoethyl) column. Which ONE of the following statements is CORRECT regarding this experiment? - A buffer of pH 6.0 would be needed to elute Protein X from the column. - Since the pI is above the physiological range, it can't be purified from a mixture of proteins. - Once Protein X is bound to the column, a high salt buffer could be used to elute it. - The protein will elute in the void volume. Which ONE of the following enzymes or chemical reagents will not cleave the following polypeptide (amino acids represented by their 1-letter code)? P-A-R-T-Y-T-I-M-E - Cyanogen Bromide - Carboxypeptidase B - Trypsin - Chymotrypsin Which of the following is not true of the reaction catalyzed by the pyruvate dehydrogenase complex? Biotin participates in the decarboxylation. Both NAD+ and a flavin nucleotide act as electron carriers. The reaction occurs in the mitochondrial matrix. The substrate is held by the lipoyl-lysine "swinging arm." Two different cofactors containing —SH groups participate. Biotin participates in the decarboxylation. Which of the below is not required for the oxidative decarboxylation of pyruvate to form acetyl- CoA? ATP CoA-SH FAD Lipoic acid NAD+ ATP Which combination of cofactors is involved in the conversion of pyruvate to acetyl-CoA? TPP, lipoic acid, and NAD+ Which of the following statements about the oxidative decarboxylation of pyruvate in aerobic conditions in animal cells is correct? One of the products of the reactions of the pyruvate dehydrogenase complex is a thioester of acetate. The methyl (—CH3) group is eliminated as CO2. The process occurs in the cytosolic compartment of the cell. The pyruvate dehydrogenase complex uses all of the following as cofactors: NAD+, lipoic acid, pyridoxal phosphate (PLP), and FAD. The reaction is so important to energy production that pyruvate dehydrogenase operates at full speed under all conditions. One of the products of the reactions of the pyruvate dehydrogenase complex is a thioester of acetate. Glucose labeled with 14C in C-3 and C-4 is completely converted to acetyl-CoA via glycolysis and the pyruvate dehydrogenase complex. What percentage of the acetyl-CoA molecules formed will be labeled with 14C, and in which position of the acetyl moiety will the 14C label be found? No label will be found in the acetyl-CoA molecules. Which of the following is not true of the citric acid cycle? All enzymes of the cycle are located in the cytoplasm, except succinate dehydrogenase, which is bound to the inner mitochondrial membrane. In the presence of malonate, one would expect succinate to accumulate. Oxaloacetate is used as a substrate but is not consumed in the cycle. Succinate dehydrogenase channels electrons directly into the electron transfer chain. The condensing enzyme is subject to allosteric regulation by ATP and NADH. All enzymes of the cycle are located in the cytoplasm, except succinate dehydrogenase, which is bound to the inner mitochondrial membrane. Acetyl-CoA labeled with 14C in both of its acetate carbon atoms is incubated with unlabeled oxaloacetate and a crude tissue preparation capable of carrying out the reactions of the citric acid cycle. After one turn of the cycle, oxaloacetate would have 14C in: all four carbon atoms. Malonate is a competitive inhibitor of succinate dehydrogenase. If malonate is added to a mitochondrial preparation that is oxidizing pyruvate as a substrate, which of the following compounds would you expect to decrease in concentration? Citrate Fumarate Isocitrate Pyruvate Succinate Fumarate Which of the following is not an intermediate of the citric acid cycle? Acetyl-CoA Citrate Oxaloacetate Succinyl-CoA α-Ketoglutarate Acetyl-CoA In mammals, each of the following occurs during the citric acid cycle except: formation of α-ketoglutarate. generation of NADH and FADH2. metabolism of acetate to carbon dioxide and water. net synthesis of oxaloacetate from acetyl-CoA. oxidation of acetyl-CoA. net synthesis of oxaloacetate from acetyl-CoA. Oxaloacetate uniformly labeled with 14C (i.e., with equal amounts of 14C in each of its carbon atoms) is condensed with unlabeled acetyl-CoA. After a single pass through the citric acid cycle back to oxaloacetate, what fraction of the original radioactivity will be found in the oxaloacetate? 1/2 Conversion of 1 mol of acetyl-CoA to 2 mol of CO2 and CoA via the citric acid cycle results in the net production of: 1 mol of FADH2. Which one of the following is not associated with the oxidation of substrates by the citric acid cycle? All of the below are involved. CO2 production Flavin reduction Lipoic acid present in some of the enzyme systems Pyridine nucleotide oxidation Pyridine nucleotide oxidation The two moles of CO2 produced in the first turn of the citric acid cycle have their origin in the: two carboxyl groups derived from oxaloacetate. The oxidative decarboxylation of α-ketoglutarate proceeds by means of multistep reactions in which all but one of the following cofactors are required. Which one is not required? ATP Coenzyme A Lipoic acid NAD+ Thiamine pyrophosphate ATP The reaction of the citric acid cycle that is most similar to the pyruvate dehydrogenase complex- catalyzed conversion of pyruvate to acetyl-CoA is the conversion of: α-ketoglutarate to succinyl-CoA. Which one of the following enzymatic activities would be decreased by thiamine deficiency? α-Ketoglutarate dehydrogenase complex The reaction of the citric acid cycle that produces an ATP equivalent (in the form of GTP) by substrate level phosphorylation is the conversion of: succinyl-CoA to succinate. The standard reduction potentials (E'°) for the following half reactions are given. Fumarate + 2H+ + 2e- → succinate E'° = +0.031 V FAD + 2H+ + 2e- → FADH2 E'° = -0.219 V If succinate, fumarate, FAD, and FADH2, all at l M concentrations, were mixed together in the presence of succinate dehydrogenase, which of the following would happen initially? Fumarate and succinate would become oxidized; FAD and FADH2 would become reduced. Fumarate would become reduced; FADH2 would become oxidized. No reaction would occur because all reactants and products are already at their standard concentrations. Succinate would become oxidized; FAD would become reduced. Succinate would become oxidized; FADH2 would be unchanged because it is a cofactor, not a substrate. Fumarate would become reduced; FADH2 would become oxidized. For the following reaction, ΔG'° = 29.7 kJ/mol. L-Malate + NAD+ → oxaloacetate + NADH + H+ The reaction as written: may occur in cells at certain concentrations of substrate and product. All of the oxidative steps of the citric acid cycle are linked to the reduction of NAD+ except the reaction catalyzed by: succinate dehydrogenase. Which of the following cofactors is required for the conversion of succinate to fumarate in the citric acid cycle? ATP Biotin FAD NAD+ NADP+ FAD In the citric acid cycle, a flavin coenzyme is required for: oxidation of succinate. Which of the following intermediates of the citric acid cycle is prochiral? Citrate Isocitrate Malate Oxaloacetate Succinate Citrate Anaplerotic reactions . produce oxaloacetate and malate to maintain constant levels of citric acid cycle intermediates produce biotin needed by pyruvate carboxylase recycle pantothenate used to make CoA produce pyruvate and citrate to maintain constant levels of citric acid cycle intermediates All of the above produce oxaloacetate and malate to maintain constant levels of citric acid cycle intermediates Intermediates in the citric acid cycle are used as precursors in the biosynthesis of: amino acids. nucleotides. fatty acids. sterols. All of the above All of the above The conversion of 1 mol of pyruvate to 3 mol of CO2 via pyruvate dehydrogenase and the citric acid cycle also yields mol of NADH, mol of FADH2, and mol of ATP (or GTP). 4; 1; 1 During the reaction of pyruvate carboxylase, CO2 is covalently attached to all the following except: phosphate. biotin. pyruvate. lysine. All of the above lysine Entry of acetyl-CoA into the citric acid cycle is decreased when: the ratio of [ATP]/[ADP] is high. Citrate synthase and the NAD+-specific isocitrate dehydrogenase are two key regulatory enzymes of the citric acid cycle. These enzymes are inhibited by: ATP and/or NADH. During seed germination, the glyoxylate pathway is important to plants because it enables them to: carry out the net synthesis of glucose from acetyl-CoA. A function of the glyoxylate cycle, in conjunction with the citric acid cycle, is to accomplish the: A) complete oxidation of acetyl-CoA to CO2 plus reduced coenzymes. B) net conversion of lipid to carbohydrate. C) net synthesis of four-carbon dicarboxylic acids from acetyl-CoA. D) net synthesis of long-chain fatty acids from citric acid cycle intermediates. E) Both B and C are correct. E) Both B and C are correct. The glyoxylate cycle is: a means of using acetate for both energy and biosynthetic precursors. The citric acid cycle begins with the condensation of acetyl-CoA with oxaloacetate. Describe three possible sources for the acetyl-CoA. Acetyl-CoA is produced by (1) the pyruvate dehydrogenase complex, (2) β oxidation of fatty acids, or (3) degradation of certain amino acids. Briefly describe the relationship of the pyruvate dehydrogenase complex reaction to glycolysis and the citric acid cycle. The pyruvate dehydrogenase complex converts pyruvate, the product of glycolysis, into acetyl- CoA, the starting material for the citric acid cycle. Describe the enzymes, cofactors, intermediates, and products the pyruvate dehydrogenase complex. The pyruvate dehydrogenase complex consists of multiple copies of each of three enzymes. The first enzyme to act is pyruvate dehydrogenase (E1), which converts pyruvate to CO2 and the hydroxyethyl derivative of thiamine pyrophosphate (TPP). The same enzyme then oxidizes the hydroxyethyl group to an acetyl group attached to enzyme-bound lipoate through a thioester linkage. The second enzyme, dihydrolipoyl transacetylase (E2), transfers the acetyl group to coenzyme A, forming acetyl-CoA. The third enzyme, dihydrolipoyl dehydrogenase (E3), oxidizes the dihydro-lipoate to its disulfide form, passing the electrons through FAD to NAD+. (See Fig. 16-6.) Suppose you found an overly high level of pyruvate in a patient's blood and urine. One possible cause is a genetic defect in the enzyme pyruvate dehydrogenase, but another plausible cause is a specific vitamin deficiency. Explain what vitamin might be deficient in the diet, and why that would account for high levels of pyruvate to be excreted in the urine. How would you determine which explanation is correct? The most likely explanation is that the patient has a deficiency of thiamine, without which the cell cannot make thiamine pyrophosphate, the cofactor for pyruvate dehydrogenase. The inability to oxidize pyruvate produced by glycolysis to acetyl-CoA would lead to accumulation of pyruvate in blood and urine. The most direct test for this deficiency is to feed a diet supplemented with thiamine and determine whether urinary pyruvate levels fall. Match the cofactors below with their roles in the pyruvate dehydrogenase complex reaction. Cofactors: A. Coenzyme A (CoA-SH) B. NAD+ C. Thiamine pyrophosphate (TPP) D. FAD E. Lipoic acid in oxidized form Roles: Attacks and attaches to the central carbon in pyruvate Oxidizes FADH2 Accepts the acetyl group from reduced lipoic acid Oxidizes the reduced form of lipoic acid Initial electron acceptor in oxidation of pyruvate. C; B; A; D; E Two of the steps in the oxidative decarboxylation of pyruvate to acetyl-CoA do not involve the three carbons of pyruvate, yet are essential to the operation of the pyruvate dehydrogenase complex. Explain. The two steps catalyzed by dihydrolipoyl dehydrogenase (E3) are required to regenerate the oxidized form of lipoate, bound to dihydrolipoyl transacetylase, from the dihydrolipoyl (reduced) form produced in the oxidation of pyruvate. First, FAD is reduced to FADH2 to reoxidize the dihydrolipoate, then NAD+ is reduced to NADH to reoxidize the FADH2 to complete the reaction. What is the function of FAD in the pyruvate dehydrogenase complex? How is it regenerated? FAD serves as the electron acceptor in the re-oxidation of the cofactor dihydrolipoate. It is converted to FADH2 by this reaction and is regenerated by the passage of electrons to NAD+. The human disease beriberi is caused by a deficiency of thiamine in the diet. People with severe beriberi have higher than normal levels of pyruvate in their blood and urine. Explain this observation in terms of specific enzymatic reaction(s). Thiamine is essential for the synthesis of the cofactor thiamine pyrophosphate (TPP). Without this cofactor the pyruvate dehydrogenase complex cannot convert pyruvate into acetyl-CoA, so the pyruvate produced by glycolysis accumulates. There are few, if any, humans with defects in the enzymes of the citric acid cycle. Explain this observation in terms of the role of the citric acid cycle. The citric acid cycle is central to all aerobic energy-yielding metabolisms and also plays a critical role in biosynthetic reactions by providing precursors. Mutations in the enzymes of the citric acid cycle are likely to be lethal during fetal development. Preparation of an extract of muscle results in a dramatic decrease in the concentration of citric acid cycle intermediates compared to their concentrations in the tissue. However, in 1935, Szent-Gyorgi showed that the production of CO2 by the extract increased when succinate was added. In fact, for every mole of succinate added, many extra moles of CO2 were produced. Explain this effect in terms of the known catabolic pathways. Succinate is an intermediate in the citric acid cycle that is not consumed but is regenerated by the operation of the cycle. By adding succinate to an extract that is depleted in citric acid cycle intermediates, these intermediates are replenished and the cycle can resume operating, oxidizing acetyl-CoA to CO2. Draw the citric acid cycle from isocitrate to fumarate only, showing and naming each intermediate. Show where high-energy phosphate compounds or reduced electron carriers are produced or consumed, and name the enzyme that catalyzes each step. This part of the citric acid cycle includes the reactions catalyzed by isocitrate dehydrogenase, the α-ketoglutarate dehydrogenase complex, succinyl-CoA synthetase, and succinate dehydrogenase. (See Fig. 16-7.) Show the three reactions in the citric acid cycle in which NADH is produced, including the structures. None of these reactions involves molecular oxygen (O2), but all three reactions are strongly inhibited by anaerobic conditions; explain why. NADH is produced in the reactions catalyzed by isocitrate dehydrogenase, the α-ketoglutarate dehydrogenase complex, and malate dehydrogenase. These reactions are indirectly dependent on the presence of O2 because the NADH produced in the reactions is normally recycled to NAD+ by passage of electrons from NADH through the respiratory chain to O2. (See also Fig. 16-7.) At what point in the citric acid cycle do the methyl carbon from acetyl-CoA and the carbonyl carbon from oxaloacetate become chemically equivalent? This happens with the formation of succinate. Show the reactions by which α-ketoglutarate is converted to malate in the citric acid cycle. The reactions are those catalyzed by the α-ketoglutarate dehydrogenase complex, succinyl-CoA synthetase, succinate dehydrogenase, and fumarase. (See Fig. 16-7.) Show the steps of the citric acid cycle in which a six-carbon compound is converted into the first four-carbon intermediate in the path. For each step, show structures of substrate and product, name the enzyme responsible, and show where cofactors participate. The reactions are those catalyzed by isocitrate dehydrogenase, the α-ketoglutarate dehydrogenase complex, and succinyl-CoA synthetase. (See Fig. 16-7.) Show the structures of the reactants and products for two of the four redox reactions in the citric acid cycle. Indicate where any cofactors participate, and label the reactants, products, and cofactors as oxidants or reductants in the reaction. The four oxidation-reduction reactions are those catalyzed by isocitrate dehydrogenase, the α- ketoglutarate dehydrogenase complex, succinate dehydrogenase, and malate dehydrogenase. (See Fig. 16-7.) For isocitrate dehydrogenase, isocitrate is the reductant (i.e. it becomes oxidized), NAD+ is the oxidant (i.e. it becomes reduced), and a divalent metal is required for this reaction. For the α-ketoglutarate dehydrogenase complex, α-ketoglutarate is the reductant, NAD+ is the oxidant, and the enzyme requires the same menagerie of cofactors as does pyruvate dehydrogenase (TPP, lipoyllysine, FAD). For malate dehydrogenase, malate is the reductant, NAD+ is the oxidant, and no cofactors are required. Show the steps of the citric acid cycle from succinyl-CoA to oxaloacetate only. For each step, show structures of substrate and product, name the enzyme responsible, and show where cofactors participate. These are the steps catalyzed by succinyl-CoA synthetase, succinate dehydrogenase, fumarase, and malate dehydrogenase. (See Fig. 16-7) Explain why fluorocitrate, a potent inhibitor of the enzyme aconitase, is a deadly poison. By inhibiting aconitase, fluorocitrate prevents the citric acid cycle from operating. This prevents the oxidation of acetyl-CoA and dramatically reduces the yield of ATP from carbohydrate and lipid catabolism. The resulting drop in ATP levels is lethal. The citric acid cycle is frequently described as the major pathway of aerobic catabolism, which means that it is an oxygen-dependent degradative process. However, none of the reactions of the cycle directly involves oxygen as a reactant. Why is the pathway oxygen-dependent? The citric acid cycle produces NADH, which normally is recycled by passage of electrons from NADH to O2 via the respiratory chain. With no O2 to accept electrons from NADH, the accumulation of NADH effectively stops the citric acid cycle. In the citric acid cycle, a five-carbon compound is decarboxylated to yield an activated four- carbon compound. Show the substrate and product in this step, and indicate where any cofactor(s) participate(s). The oxidation of α-ketoglutarate to succinyl-CoA involves five cofactors: lipoate, thiamine pyrophosphate (TPP), FAD, NAD+, and CoA-SH. CO2 is produced in two reactions in the citric acid cycle. For each of these reactions, name and show the structures of reactant and product, name the enzyme, and show how any cofactors participate. See the isocitrate dehydrogenase and α-ketoglutarate dehydrogenase reactions. (See also Fig. 16- 6.) In which reaction of the citric acid cycle does substrate-level phosphorylation occur? Substrate-level phosphorylation of GDP to GTP occurs in the succinyl-CoA synthetase reaction in which succinyl-CoA is converted to succinate during the citric acid cycle. Explain in quantitative terms the circumstances under which the following reaction can proceed. L-Malate + NAD+ → oxaloacetate + NADH + H+ ΔG'° = +29.7 kJ/mol A reaction for which ΔG'° is positive can proceed under conditions in which the actual ΔG is negative. From the relationship ΔG = ΔG'° + RT ln [product], [reactant] it is clear that if the concentration of product is kept very low (e.g., by its removal in a subsequent metabolic step), the logarithmic term becomes negative and the actual ΔG can then have a negative value. (See also Chapter 13.) You are in charge of genetically engineering a new bacterium that will derive all of its ATP from sunlight by photosynthesis. Will you put the enzymes of the citric acid cycle in this organism? Briefly explain why or why not. Yes; even though the citric acid cycle is not needed for catabolic reactions in this organism, the enzymes of the cycle are still essential. They produce precursors of amino acids (such as α-keto- glutarate and oxaloacetate), of heme (succinyl-CoA), and of a variety of other essential products. Match the cofactor with its function in the citric acid cycle. A given function may be used more than once or not at all. Cofactor Function (a) NAD+/NADH (1) carries O2 (b) FAD/FADH2 (2) carries small carbon-containing molecules (c) CoA (3) carries e- (d) thiamine (4) carries small nitrogen-containing molecules (e) biotin (a)-(3); (b)-(3); (c)-(2); (d)-(2); (e)-(2) Germinating plant seeds can convert stored fatty acids into oxaloacetate and a variety of carbohydrates. Animals cannot synthesize significant quantities of oxaloacetate or glucose from fatty acids. What accounts for this difference? Plants use the glyoxylate cycle to convert two molecules of acetyl-CoA into one four-carbon compound (such as oxaloacetate), then use this compound to make glucose (gluconeogenesis). In animals that lack the glyoxylate cycle, each acetyl group that enters the citric acid cycle yields two CO2, allowing no net conversion of acetyl groups into oxaloacetate. Learn More You can also click on terms or definitions to blur or reveal them UNIT 6, L1 For metabolism in multicellular organisms to proceed efficiently, it is important that the final products be gases, water, or both. Why? To maintain metabolism in a steady state, there must be no possibility of a build-up of final products. The easiest disposal products are the multicellular organism's universal solvent, gases, or both. Look at the two starred reactions in the lesson just covered (the redox reactions involving NADH and O2). Compare the tendency of NADH to donate electrons and the tendency of oxygen to accept them. If NADH and oxygen are mixed, will the electrons stay with NADH or go to oxygen? Explain. If NADH and oxygen are mixed, electrons will be transferred from NADH to oxygen with the release of considerable energy: NADH + ADH + ½ O2 + H+ ↔↔ NAD+ + H2O εo′ = 1.13 V What structural feature do the "high‑energy" compounds ATP, FADH2, and NADH share with acetyl‑CoA? The "high‑energy" compounds share an ADP unit or, in the case of acetyl‑CoA, a closely related derivative. Explain the meaning of the term "metabolic pathway" - is a sequence of enzyme - catalyzed chemical reactions within a cell that converts a starting molecule (like glucose) into a final product (like pyruvate) - each step involved a small chemical transformation, and the entire pathways functions to either break down molecule to release energy or build larger using energy List the principle characteristics of metabolic pathways and explain the purpose of each of the two major types of pathways Principle characteristics: Composed of a series of enzyme catalyzed reactions Often maintained in a steady state, not equilibrium Controlled by: Rate of substrate entry (glucose into the cell) Rate of product (pyruvate) Diversion of enzyme Feedback mechanisms (product inhibition of earlier steps ) Include both reversible and irreversible reactions Some reactions serves as regulatory control points (typically irreversible) Two Major Types of Pathways: Catabolic pathways: Break down large molecules into smaller ones Release energy often captured as ATP Example: glucose (glucose - pyruvate) Anabolic pathways: Build larger molecules from smaller precursors Consume energy, usually in the form of ATP or NADPH Example: protein synthesis from amino acids Identify the types of chemical reactions that are important in biochemistry. Oxidation reduction: Transfers of pair of electrons (NADH donates electrons; O2 accepts them) Central to energy production Elimination, isomerization and rearrangement: Elimination: removal of small molecules (H2O, NH3) Isomerization: internal shift of atoms (hydrogen) rearrangement : breaking and reforming carbon-carbon bonds Group transfer: Transfer of groups like phosphoryl, acyl, or glycosyl from one molecule to another Often involved energy carriers like ATP Define "irreversible metabolism" and "steady state" as they apply to metabolism Irreversible metabolism refers to reactions within a metabolic pathway that occur far from equilibrium and have a large negative deltaG. These reactions proceed in only one direction and typically serve as regulatory control points Steady state in metabolism means the concentrations of reactants and products remain constant over time, even though reactions are continuously occurring Unlike equilibrium (where deltaG = 0 and no net reaction occurs), steady state involved continuous input and output of materials (like water in a bucket with a hole and running tap) Explain the source of "energy" in high energy compounds High energy compounds (ATP, phosphocreatine) release energy upon hydrolysis due to: 1. resonance stabilization: products have more resonance forms than reactants 2. electrostatic repulsion: breaking high repulsion bonds lowers the energy state 3. entropy increase (delta S): products are more disordered, favouring energy release The more negative the standard free energy change (delta G) of hydrolysis the greater the energy content of the original compounds Relate the metabolic pathways in eukaryotes cells to the specific cellular location of these metabolic events Metabolic events are compartmentalized in eukaryotic cells: Glycolysis: occurs in the cytoplasm Tricarboxylic acid (TCA) cycle: takes place in the mitochondrial matrix Electron transport chain (ETC): located on the inner mitochondrial membrane Glucose metabolism: happens in all cells, but efficiency varies (high in muscle cells, lower in gut epithelial cells) UNIT 6, L2 Explain the difference between standard free energies (ΔGo′) and physiological free energies (ΔG ). The standard free energy values (ΔGo′) indicate how much product relative to reactant there will be at equilibrium A “physiological free energy change” (ΔG) is the amount of energy released when a reaction is considered inside the cell (at steady state) Define "substrate‑level phosphorylation." results in the formation of ATP or GTP by conversion of a higher energy substrate (whether phosphate group attached or not) into lower energy product and a using some of the released chemical energy, the Gibbs free energy, to transfer a phosphoryl (PO3) group to ADP or GDP from another phosphorylated compound Add up the ten physiological free energy values for the ten glycolysis reactions in erythrocytes (see the table in the commentary above). What is the overall ΔG for glycolysis? Why can the reaction never come to equilibrium in vivo? ΔG = −74.0 kJ/mol for the overall glycolytic process. This large negative value indicates that the overall reaction is spontaneous (i.e., the final product, pyruvate, is being removed fast enough, and glucose is being added constantly enough), so that there is no back reaction: Pyruvate → Glucose Glycolysis is inhibited by iodoacetic acid through inactivation of the enzyme glyceraldehyde‑3‑phosphate dehydrogenase (GAPDH). As a result, there is an accumulation of fructose‑1,6‑biphosphate. Why is this product more prevalent than glyceraldehyde‑3‑phosphate and dihydroxyacetone phosphate, the products that form immediately preceding GAPDH? The reaction that converts fructose 1,6‑bisphosphate to glyceraldehyde‑3‑phosphate and dihydroxyacetone phosphate is near equilibrium in vivo (i.e., ΔG near zero), while the preceding reaction producing fructose 1,6‑bisphosphate is not. Therefore it is F1,6BP that will accumulate, not glyceraldehyde‑3‑phosphate and dihydroxyacetone phosphate. The "Pasteur effect" is the dramatic decrease in glucose consumption when oxygen is introduced to an anaerobic fermentation broth. Why do the yeast cells use less glucose after oxygen is introduced? How much less glucose do they use after oxygen is introduced? The introduction of oxygen allows yeast to convert from anaerobic to aerobic metabolism. Since aerobic metabolism provides more ATP, the amount of glucose that must be used to nourish the yeast is much less. Approximately 6% of the glucose metabolized anaerobically is needed to provide the same amount of energy under aerobic conditions. Why do you get hot when you exercise? PEP is a very high‑energy compound. The reaction that converts PEP to pyruvate is so highly energetically favourable (very negative ΔG) that there is almost enough energy in PEP to stimulate production of a second ATP through substrate level phosphorylation, but it is not used. The excess energy is lost as heat. What are the three enzymes that are regulated in glycolysis? How does AMP affect glycolysis? The three enzymes that are regulated in glycolysis are: hexokinase, phosphofructokinase (PFK), and pyruvate kinase. High amounts of AMP activate PFK and pyruvate kinase, which stimulate glycolysis because ATP is needed. List the possible uses of pyruvate. The possible uses of pyruvate are: - ATP production and NAD+ regeneration through TCA cycle and electron transport - glucose synthesis by gluconeogenesis - ATP production and NAD+ generation and lactate or ethanol production by fermentation - alanine synthesis - oxaloacetate synthesis What does the liver do with the lactate that is produced during heavy exercise? During heavy exercise, the body needs ATP and generates lactate through anaerobic respiration faster than the blood can deliver oxygen to continue through aerobic respiration. Muscle cells cannot use the lactate produced anaerobically during exercise, so the lactate is dumped into the blood. The liver converts the lactate to pyruvate in the Cori Cycle. The enzyme lactate dehydrogenase catalyzes this conversion. The pyruvate is then used to make glucose by gluconeogenesis in the liver, and can go back into the blood to be taken up by muscles and used for energy. Demonstrate an understanding of the reactions in glycolysis Glycolysis is a series of 10 enzyme controlled steps that convert glucose (6C) into 2 pyruvate (3C each) molecules: It occurs in the cytosol, is anaerobic, and produces: 2 ATP (net) 2 NADH 2 H2O 2H+ Reactions are divided into: Energy investment phase (uses 2 ATP) Energy payoff phase (produces 4 ATP + 2 NADH) Explain the chemical strategy by which glycolysis yields a "profitL of two ATP molecules per glucose molecule oxidized" Two ATP are consumed in early steps: Glucose - glucose-6-phosphate Fructose-6-phosphate - fructose-1,6-bisphosphate Four ATP are produced later through substrate level phosphorylation: 1,3-bisphosphoglycerate _ 3-phosphoglycerate PEP - pyruvate Net gain = 4-2 = 2 ATP per glucose Use standard free energy (delta G') and physiological free energies (delta g) to determine control points in multi enzyme process Control porints are steps with large, negative ΔG values under physiological conditions - far from equilibrium In glycolysis, the three major control points are: Hexokinase Phosphofructokinase (PFK) Pyruvate kinase These are irreversible reactions, their regulation ensures unidirectional flow of metabolites Name and describe two processes for anaerobic replenishment of NAD+ Lactic acid fermentation (in animals): Pyruvate + NADH - lactate + NAD+ (via lactate dehydrogenase) Alcoholic fermentation (in yeast): Pyruvate - acetaldehyde - ethanol NADH - NAD+ in the conversion of acetaldehyde to ethanol Both pathways regenerate NAD+, allowing glycolysis to continue in anaerobic conditions Name the enzymes, reactants and products for three irreversible reactions of glycolysis Step Enzyme Reactant → Product 1. Hexokinase step Hexokinase Glucose + ATP → Glucose-6-phosphate + ADP 3. PFK step Phosphofructokinase (PFK) Fructose-6-phosphate + ATP → Fructose-1,6-bisphosphate + ADP 10. Pyruvate kinase step Pyruvate kinase Phosphoenolpyruvate + ADP → Pyruvate + ATP Write the overall reaction for glycolysis Glucose + 2 NAD+ + 2ADP + 2Pi - 2 pyruvate + 2NADH + 2 ATP + 2H2O + 2H+ Compare the pathways of glycolysis and gluconeogenesis and discuss the regulation of these pathways Glycolysis: catabolic , breaks down glucose - pyruvate - energy (ATP) Active when ATP is low, AMP is high Key enzymes inhibited by ATP, activated by AMP/F-2, 6-BP Gluconeogenesis: Anabolic, synthesizes glucose from pyruvate Active when ATP is high Uses 7 shared enzymes (ΔG ≈ 0), and 4 unique enzymes to bypass irreversible glycolysis steps Reciprocal regulation ensures: No futile cycling When one pathway is active, the other inhibited Explain the basis of the cori cycle During intense activity, muscles perform anaerobic glycolysis, producing lactate Lactate travels to the liver, where it is: Converted back to pyruvate Then used in gluconeogenesis to regenerate glucose The glucose returns to muscle for energy use This CORI cycle helps clear lactic acid and maintain glucose supply during recovery UNIT 6, L3 Can one get a net synthesis of oxaloacetate if one adds acetyl‑CoA to a system that contains only the enzymes and intermediates of the citric acid cycle? (Consider one complete cycle.) No, one cannot get a net synthesis of oxaloacetate in these circumstances. Acetyl‑CoA is a 2‑carbon species and two molecules of CO2 are given off for each turn of the cycle. Therefore no net synthesis is possible. In fact, this is the point of the citric acid cycle. All the atoms of glucose are discarded and the glucose energy is conserved in one molecule of GTP and in high‑energy electrons. What is the purpose of the production of NADH and FADH2 in the citric acid cycle (CAC or TCA)? The production of NADH and FADH2 is one of the most important features of the CAC. These compounds are needed to drive the electron transport chain for energy (ATP) production. In general, how is the CAC connected to many other metabolic pathways? What compound acts as the link to these pathways? Acetyl‑CoA is central in linking glycolysis with the citric acid cycle, but many other metabolic pathways can be fed through the CAC and on to electron transport for energy production. Fatty acids and amino acids, as well as carbohydrates, can be metabolized to acetyl‑CoA. The bulk of the ATP molecules that result from "metabolism" come from the processing of acetyl‑CoA through the citric acid cycle, and then into the electron transport chain. What two enzymes are present in the glyoxylate cycle that animals lack? The two enzymes present in the glyoxylate cycle that animals lack are isocitrate lyase and malate synthase. Compare the overall outcomes of the CAC and the glyoxylate cycles. The CAC and the glyxoylate cycle differ in that the CAC produces 1 oxaloacetate per turn of the cycle and the glyoxylate cycle produces 2. This means that there is no net production of oxaloacetate in the CAC; the one that is made balances out the one used for the cycle. The extra one produced by the glyoxylate cycle in plants and bacteria means that, unlike animals, these organisms can use the oxaloacetate to make glucose and other molecules. Plants and bacteria can turn acetyl‑CoA into glucose, while animals cannot. It also means that plants and bacteria can turn acetyl‑CoA from fat into glucose but animals cannot. The advantage of the CAC in animals, though, is the higher production of NADH and FADH2. One turn of the glyoxylate cycle produces 1 NADH and 1 FADH2, whereas one turn of the CAC results in 3 NADH, 1 FADH2, and 1 GTP. This makes sense considering the high energy requirements of animals. Why is the glyoxylate cycle important for plants, fungi, protists, and bacteria? Why would the CAC be more important for animals? The higher energy output of the CAC for animals provides the requirements for the production of higher amounts of ATP through the electron transport chain, which is needed for the mobility of animals. The extra oxaloacetate produced through the glyoxylate cycle in plants, bacteria, and protists is necessary to produce carbohydrate for structure and storage, which are necessary functions for these organisms. Explain the importance of the regeneration of oxaloacetate to TCA cycle function Oxaloacetate is four carbon molecule that combines with acetyl-CoA to form citrate the first step of the TCA cycle The cycle is cyclic, oxaloacetate must be regenerated at the end of each turn to allow the cycle to continue Without it, acetyl-CoA cannot enter the cycle, halting energy production, this regeneration ensures the TCAcycle remains a continuous process for metabolizing fuels and producing high energy compounds Explain how the energy released when glucose is oxidized through the TCA cycle is chemically "stored" As glucose is oxidized, it is broken down into pyruvae (via glycolysis) and then to acetyl-CoA which enter the TCA cycle. In the TCA cycle, the chemical energy is stored in: 3 NADH 1 FADH2 1 GTP (functionally equivalent to ATP) These high energy molecules store the energy released from the oxidation of glucose and carry electrons to the electron transport chain (ETC) where the majority of ATP is produced via oxidative phosphorylation Explain why NADH and FADH2 are considered high energy compounds NADH and FADH2 are considered high energy because they carry pairs of high energy electrons These electrons are in a reduced state, meaning they can be donated to the electron transport chain Their aromatic ring structure (especially NAD+) are resonance stabilized and the reduced forms (NADH/FADH2) are less stable, thu they have a strong drive to donate electrons, releasing energy in the process List chemical transformations that occur with one turn of he TCA cycle Per one turn of the TCA cycle (starting with one acetyl-CoA): acetyl=CoA (2C) + oxaloacetate (4C) - CITRATE (6c) Series of transformations involving dehydration, hydration, and decarboxylation reactions lead to: 2 CO2 molecules released 3 NAD+ - 3 NADH 1 FAD - 1 FADH2 2 GDP + Pi - 1 GTP Oxaloacetate is regenerated Describe the metabolic important of acetyl CoA acetyl-CoA is a central metabolic intermediate: It links glycolysis, fatty acid oxidation, and amino acid catabolism to the TCA cycle It is the entry point for carbon into the TCA cycle While animals use it primarily for energy production , plants, fungi, and bacteria can also use it to synthesize glucose via the glyoxylate cycle Identify the enzymes involved in the regulation of the TCA cycle TCA cycle is regulated at three main enzymes, which are likely: Citrate synthase Isocitrate dehydrogenase Alpha ketoglutarate dehydrogenase These enzymes are rate limiting and responsive to substrate availability, product accumulation, and energy status of the cell Identify the mechanisms by which the TCA cycle is regulated TCA cycle is regulated through: Allosteric inhibition/activation by intermediates and products (NADH inhibits key enzymes) Energy status: High levels of ATP and NADH signal energy sufficiency and inhibit the cycle, while high ADP or NAD+ stimulate it Substrate availability: levels of acetyl-CoA, oxaloacetate, and other intermediates impact flux through the cycle Explain how the functioning of the electron transport chain keeps TCA cycle produces NADH and FADH2, which must be oxidized bacl to NAD+ and FAD for the cycle to continue This oxidation occurs in the eTC Without the ETC, NADH and FADH2 would accumulate and NAD+/FAD would be depleted, halting the TCA cycle Therefore the ETC maintains redox balance, enabling continuous operation of the TCA cycle and sustained ATP production UNIT 6, L4 Why is acetyl‑CoA such an important molecule? Name three pathways that require this molecule. Acetyl‑CoA is an important molecule, because it is used in many reactions and links many different metabolic pathways. The function of this molecule is to move the C atoms in the acetyl group to the CAC and then transport electrons for ATP production. Three pathways that require this molecule are: fatty acid oxidation and reduction, pyruvate oxidation, the citric acid cycle (CAC), (also amino acid anabolism and catabolism, ketone body metabolism). What type of bond is the high energy bond in acetyl‑CoA? The high energy bond in acetyl‑CoA is a thioester bond. Hydrolysis of this bond is highly exergonic (−31.5 kJ). List three functions of cholesterol in the body. The three functions of cholesterol in the body are: cell membrane structure, precursor of steroid hormones, precursor of vitamin D (also bile acid precursor). What is the name of the pathway involved in the production of cholesterol? Why is this pathway regulated? How do levels of AMP affect the regulation of this pathway? The pathway involved in cholesterol synthesis is the isoprenoid pathway. Because it requires a large energy input, this pathway is regulated so that it will not run unless it is needed. When concentrations of AMP are high, the cell doesn't have a lot of energy to produce cholesterol. An AMP‑activated protein kinase inhibits HMG‑CoA reductase by phosphorylating it, which inhibits HMG‑CoA reductase and halts cholesterol production. Why do you think HMG‑CoA reductase is an ideal target for cholesterol‑reducing medications? HMG‑CoA reductase is an ideal target for cholesterol‑reducing medications because it is a key enzyme in the synthesis of cholesterol and it is regulated in vivo. Statins are used clinically for treatment. They are fungal products that act as a competitive inhibitor of HMG‑CoA reductase. Why are bile acids important in metabolism? What molecule is converted to bile acids? Insoluble products such as dietary fibre, and waste products such as heme degradation products, must be efficiently eliminated by combination with a detergent. A detergent molecule is one that combines with a non‑soluble molecule, giving a complex which can be suspended in solution rather than clumping with like insoluble molecules. The bile salts, cholic acid and deoxycholic acid, are the major biological detergents. Cholesterol is the precursor of both cholic acid and deoxycholic acid. Why do those on a strict vegetarian diet rarely suffer from diet-induced hypercholesterolemia? Cholesterol is not found in plants; therefore, strict vegetarians rarely suffer from diet‑induced hypercholesterolemia. Demonstrate an understanding of the role of acetyl-CoA in metabolism acetyl-CoA is a central metabolic intermediate involved in numerous pathways It serves primarily to transfer acetyl groups (2-carbon units) into the citric acid cycle (CAC) for oxidation and energy production Beyond energy metabolism, acetyl-CoA also plays critical roles in: Fatty acid oxidation and synthesis Pyruvate oxidation Amino acid catabolism and anabolism Ketone body production Synthesis of cholesterol Because of its centrality, acetyl-CoA links carbohydrates, lipid, and protein metabolism Describe the high energy tiolester bond in acetyl-CoA The acetyl group in acetyl-CoA is linked to coenzyme A by a thioester bond This bond is high energy and hydrolysis release -31.5kj/mol making the reaction strongly exergonic This energy drives metabolic reactions forward, such as the entry of acetyl-CoA into the CAC The thioester bond is formed between the acetyl group and the sulfhydryl group of beta- mercaptoethylamine, a component of coenzyme A Explain how cholesterol is transported and described how it is taken up by cells Cholesterol is transported through the bloodstream in lipoprotein complexes, including: LDL (low density lipoprotein): delivers cholesterol to tissues HDL (high density lipoprotein): scavenges excess cholesterol from cells and membranes chylomicrons : carry dietary (non-esterified) cholesterol Cellular uptake of cholesterol occurs primarily via LDL endocytosis: LDL receptors on the cell surface bind LDL particles and internalize them When intracellular cholesterol levels are high, LDL receptor synthesis is downregulated When cholesterol is low LDL receptor expression increases, enhancing uptake List the functions of cholesterol in the body Cholesterol is a crucial structural and functional molecule, with roles including: Components of cell membranes: maintain fluidity and integrity Precursor to steroid hormones: eg; cortisol, estrogen, and testosterone Precursor to bile acids: essential for digestion and fat absorption Precursor to vitamin D Transport and signalling roles via lipoproteins (LDL, HDL) Provide a general overview of cholesterol synthesis Begins with acetyl-CoA Two acetyl-CoA molecules form acetoacetyl-CoA, then third acetyl-CoA is added to form HMG- CoA HMG-CoA reductase converts HMG-CoA into mevalonate, a key step in the isoprenoid pathways The pathway proceeds through multiple steps to form cholesterol Demonstrate an understanding of the regulation of cholesterol synthesis Cholesterol synthesis is energy-intensive and tightly regulated: Transcriptional regulation: HMG-COA reductase gene expression increases when cholesterol is low Feedback inhibition Cholesterol inhibits HMG-CoA reductase activity when levels are sufficient Covalent modification: AMP-activated protein kinase phosphorylates and inhibits HMG-COA reductase when cell energy is low (high AMP) LDL receptor regulations: High cholesterol suppresses receptor synthesis, reducing uptake Discuss the importance of the bile acids in digestion Bile acids are synthesized from cholesterol in the liver and play a vital role in digestion: Emulsifying dietary fats in the small intestine Increasing surface area for pancreatic lipase activity Aiding in the absorption of fat soluble vitamins (ADEK) They serves as a route for cholesterol elimination, as cholesterol is converted into bile acids and excretion into the digestive tract UNIT 6, L5 Name the ketone bodies. What is the role of these molecules in metabolism? When are ketone bodies a source of energy? The ketone bodies are acetoacetate, acetone, and D‑β‑hydroxybutyrate. The role of these molecules is to provide energy when glucose levels are low. They can be converted to acetyl‑CoA for entry into the citric acid cycle (CAC) and then ATP synthesis through the electron transport chain. Acetoacetate and β‑hydroxybutyrate can be converted to acetyl‑CoA. They can both cross the blood‑brain barrier and provide energy for the brain when glucose is limiting. What is the connection between the citric acid cycle and oxaloacetate production and ketone body formation? The connection between the CAC, oxaloacetate production, and ketone body formation is that acetyl‑CoA from fatty acid breakdown enters the CAC, but oxaloacetate must be present to combine with acetyl‑CoA to form citrate. Oxaloacetate is usually regenerated each turn of the CAC, but in some cases it is used for other metabolism (drawn off) and is not present. If there is insufficient oxaloacetate, the acetyl‑CoA is converted to acetoacetate or β‑hydroxybutyrate. These ketone bodies can cross into the brain to provide energy and can be converted back into acetyl‑CoA as well. How much energy can you obtain from the oxidation of palmitate, CH3 (CH2)14COOH, in the liver under conditions of ketosis? How does this compare to the amount you can obtain when on a balanced diet providing glucogenic fuel molecules? Estimates of energy release are most easily made by comparing the number of high‑energy electron carriers (NADH and FADH2) produced. Palmitate releases eight acetyl‑CoA fragments by seven rounds of β‑oxidation. Each round of β‑oxidation also yields one NADH and one FADH2 (a total of 14 high‑energy electron pairs). This number is the same for normal and ketogenic conditions. Under normal conditions each acetyl‑CoA, via the citric acid cycle, will reduce one FAD and 3NAD+ (a total of 32 high‑energy electron pairs, since there are eight acetyl‑CoA units). This latter process is not possible under ketogenic conditions. Therefore ketogenic conditions provide ~30% of the energy that is available under normal conditions. Of course, the acetyl‑CoA units produced under ketogenic conditions are still available should conditions improve. Excess acetyl‑CoA, produced by the citric acid cycle or by fatty acid breakdown, is channelled into ketone bodies. Would it be more sensible to provide a feedback mechanism to slow down the production of acetyl‑CoA itself? Such a feedback mechanism would and would not be more sensible. Ketone bodies are produced under normal conditions and are the preferred fuel of, for example, heart tissue. The problem occurs when citric acid intermediates are in short supply and therefore the ketone bodies accumulate. There is some regulation of acetyl‑CoA production, but this molecule is at the heart of metabolism. Without it, you would die, so its synthesis is never shut down completely. It was stated that one oxaloacetate molecule must be regenerated approximately every seven turns of the citric acid cycle. Since glycerol (from triacylglycerols) can produce oxaloacetate, and there are only seven to eight acetyl‑CoAs produced per fatty acid, why are ketone bodies produced at all? It is a matter of mathematics. A triacylglycerol produces one glycerol and three fatty acids. That is, one oxaloacetate for 21-24 acetyl‑CoA units. How are ketone bodies funnelled back into central metabolism if citric acid cycle intermediates are synthesized? Acetoacetate and β‑hydroxybutyrate are reconverted to acetyl‑CoA. What are the steps of biosynthesis of fatty acids? The six steps in the biosynthesis of fatty acids are: 1. conversion of acetyl‑CoA to malonyl‑CoA. 2. conversion of malonyl‑CoA to malonyl‑ACP. 3. condensation of malonyl‑ACP with the growing chain (with cleavage of CH2). 4. reduction of a carbonyl group to an alcohol group. 5. reduction of the alcohol group to a trans‑α, β double bond. 6. reduction of the C==C bond to a CH2−−CH2 single bond. Name the ketone bodies Acetoacetate Beta-hydroxybutyrate acetone Explain the normal physiological role of ketone bodies Ketone bodies serve as an alternative energy source, especially when glucose is scarce Beta-hydroxybutyrate and acetoacetate can be converted back into acetyl-cOA which enters the citric acid cycle to produce ATP These ketone bodies are utilized by muscle tissues and the brain, particularly during fasting or carbohydrate restriction Acetoacetate also plays a regulatory role by inhibiting further lipolysis (the breakdown of fats) Describe the circumstances under which ketone bodies are synthesized, and outline the chemical reactions involved in their synthesis Circumstances: Ketone bodies are synthesized in the liver when there is excess acetyl-CoA and insufficient oxaloacetate to allow entry into the citric acid cycle This occurs during fasting, low carbohydrate diets, prolonged exercise or uncontrolled diabetes when fatty acids are broken down for energy Why oxaloacetate is insufficient: Intermediates of the citric acid cycle including oxaloacetate may be diverted for the biosynthesis of glucose amino acids and other molecules Since lipids cannot produce oxaloacetate (unlike carbohydrates), the citric acid cycle slows, and excess acetyl-CoA builds up Chemical reactions (overview): Fatty acids - acetyl-CoA (via beta oxidation) acetyl-CoA - acetoacetate (initial ketone body) Acetoacetate - beta hydroxybutyrate (via reversible reduction) Acetoacetate - acetone (via spontaneous decarboxylation) UNIT 6, L6 What biological advantage does storing major fuel reserves as fat rather than glycogen confer on nonplant species? Fats are insoluble, and so are not hydrated. Adipose cells—fat storage depots—contain droplets of triacylglycerol, much like drops of fat floating on the surface of water. Glycogen is a well‑hydrated molecule. To maintain the equivalent amount of energy reserves, a glycogen storer would be considerably heavier than a fat storer. This situation is not a good one if you are mobile and trying to escape predators. Why are fat stores in mammalian adipose tissue sources of intracellular water? The complete oxidation of lipids yields carbon dioxide and water: roughly 1 mL water per gram of fat. This relationship is a great advantage to camels, whose humps are fat depots. Hydrolysis of the fat provides some water to the animal during dry desert work. What are the benefits of using β‑oxidation to metabolize lipids? Conversion of a non‑reactive, insoluble hydrocarbon chain to a useful energy intermediate presents an immediate problem of solubility. The steps of β‑oxidation provide soluble fragments, as well as a high‑energy bond on each fragment, and an intermediate that can channel into an existing metabolic pathway (the citric acid cycle). Marathon runners are said to "hit the wall" at about 20 miles, approximately the time the runner converts from carbohydrate to fat stores for energy. Why is this process so difficult that it is called a "wall"? Fats, which are less soluble than carbohydrates, cannot be marshalled for use as quickly as can carbohydrates. The lipases in adipose tissue are soluble enzymes which must nibble at the edges of the fat droplets. As the fatty acids enter metabolism, their energy content, which is greater than that of carbohydrates, partially compensates for the slow start. This fact means that you can "get over the wall." A victim of starvation has to be fed small amounts of food and has to be very careful about introducing fat into the diet. Why? A victim of starvation must be careful because the digestive and biosynthetic enzymes are produced on demand: no food = no enzymes. It normally takes three or more days for the enzymes to be synthesized (assuming amino acids are available to do so). The fatty acid degradative and biosynthetic enzymes are more slowly synthesized. You add radiolabelled malonyl‑CoA to a cell extract which is actively synthesizing palmitate. (Each carbon of the malonyl unit is labelled by the isotope 14C.) You stop the reaction one minute later by altering the pH. Then you analyze the palmitate for radioactivity. Which of the carbons will be labelled in palmitate? 14C is carbon‑1; that is, carbon‑1 is radiolabelled, because a malonyl unit is added to the growing chain by insertion at the front. Explain how lipids are degraded to acetyl-COA in vivo Lipid degradation begins with β-oxidation, a cyclic four-step process in the mitochondria. Fatty acids are first activated by attachment to coenzyme A (CoA) and then processed through: Dehydrogenation (produces FADH₂) Hydration (adds OH group) Oxidation (converts alcohol to ketone, produces NADH) Thiolysis (cleaves off acetyl-CoA and shortens the fatty acid by 2 carbons) The shortened fatty acid then re-enters the cycle until fully converted to acetyl-CoA. Explain how fatty acid chains are synthesized in vivo Fatty acid synthesis occurs in the cytosol and is catalyzed by the fatty acid synthase complex. The process uses malonyl-ACP (from acetyl-CoA) as the building block. Each cycle adds a 2-carbon unit and involves: Formation of malonyl-ACP Condensation with the growing chain (loss of one carbon as CO₂) Reduction of carbonyl to alcohol (uses NADPH) Dehydration to form a double bond Reduction of the double bond to a single bond (uses NADPH) The chain is anchored to ACP, allowing repeated elongation until the desired chain length (often 16 or 18 carbons) is reached. Explain how lipids (which are insoluble in water) are digested, absorbed, and transported Dietary lipids are broken down in the intestine into fatty acids by enzymes. These fatty acids are transported through the bloodstream as lipid-protein complexes. Cellular uptake of lipids is receptor-mediated; receptor mutations can lead to heart disease. In Western diets, excess lipids may not be fully transported or absorbed due to their water- insolubility. Describe B-oxidation (degradation) of lipids to acetyl-CoA β-oxidation consists of four repeating steps: Dehydrogenation → forms trans-double bond and produces FADH₂ Hydration → adds an OH group at carbon 3 Oxidation → converts OH to a ketone and produces NADH Thiolytic cleavage → removes acetyl-CoA, shortens the fatty acid by 2 carbo