BCH 3303 Essentials of Biochemistry Lecture Notes PDF

BCH 3303: Essentials of Biochemistry (TR 10:00 – 11:15 AM; McKinney Humanities 2.01.44) Instructor. Syed Muhammad Usama, PhD Assistant Professor Department of Chemis...

BCH 3303: Essentials of Biochemistry (TR 10:00 – 11:15 AM; McKinney Humanities 2.01.44) Instructor. Syed Muhammad Usama, PhD Assistant Professor Department of Chemistry Contact Information. Office Hours. email: [email protected] time and location: M/W (10 – 12 PM) office phone: 210-458-5641 office: BSE 1.104C or by appointment (please email) Grader. Thomas Yost Biochemistry Major, 2025 Week 10 [email protected] Lecture 19 3rd April 2025 Schedule for the Semester  Required Material Biochemistry: A Short Course Fourth Edition  Homework Assignment Homework Assignment (Chapter 16 and 17) are due on Friday, April 4th at 11:59 PM Homework Assignment (Chapter 18 and 19) are due on Friday, April 11th at 11:59 PM  CHAPTER 18 Preparation for the Cycle  The Synthesis of Acetyl Coenzyme A from Pyruvate The synthesis of acetyl CoA from pyruvate consists of three steps: a decarboxylation, an oxidation, and the transfer to CoA.  Pyruvate Dehydrogenase Complex of E. coli Prosthetic Group: A prosthetic group is a tightly bound, non-protein molecule (cofactor) that is essential for the biological activity of an enzyme, often covalently attached and involved in the enzyme's catalytic function. Stoichiometric coenzymes function as substrates CoA and NAD+ Catalytic coenzymes like enzymes are are not permanently altered by participation in the reaction TPP, lipoic acid, FAD  1. Decarboxylation Pyruvate dehydrogenase (E1), a component of the complex, catalyzes the decarboxylation. Pyruvate combines with the ionized form of the coenzyme thiamine pyrophosphate (TPP).  2. Oxidation The hydroxyethyl group attached to TPP is oxidized to form an acetyl group while being simultaneously transferred to lipoamide, a derivative of lipoic acid. The disulfide group of lipoamide is reduced to its disulfhydryl form in this reaction. The reaction is catalyzed by E1 and yields acetyl–lipoamide.  3. Formation of Acetyl CoA E2 catalyzes the transfer of the acetyl group from acetyl–lipoamide to coenzyme A to form acetyl CoA.  4. The regeneration of Dihydrolipoamide To participate in another reaction cycle, dihydrolipoamide must be reoxidized. This reaction is catalyzed by dihydrolipoamide dehydrogenase (E3). Reactions of the Pyruvate Dehydrogenase Complex  The Pyruvate Dehydrogenase Is Regulated by Two Mechanisms The formation of acetyl CoA from pyruvate is irreversible in animal cells. Acetyl CoA has two principle fates: metabolism by the citric acid cycle or incorporation into fatty acids.  1. Phosphorylation/Dephosphorylation of Enzyme E1 Enzyme E1 is a key site of regulation. A kinase associated with the complex phosphorylates and inactivates E1. A phosphatase, also associated with the complex, removes the phosphate and thereby activates the enzyme.  2. Energy Charge during rest during physical exercise The pyruvate dehydrogenase complex is also regulated by energy charge. ATP, acetyl CoA, and NADH inhibit the complex. ADP and pyruvate stimulate the complex.  CHAPTER 19 Harvesting Electrons from the Cycle Citric Acid Cycle Glucose pyruvate c-c-c-c-c-c c-c-c pyruvate Acetyl CoA citric acid c-c-c c-c c-c-c-c-c-c CO2 CO2 Oxalacetate c-c-c-c Citric Acid Cycle Glucose pyruvate c-c-c-c-c-c c-c-c pyruvate Acetyl CoA citric acid c-c-c c-c c-c-c-c-c-c CO2 CO2 Oxalacetate c-c-c-c Citric Acid Cycle Glucose pyruvate c-c-c-c-c-c c-c-c pyruvate Acetyl CoA citric acid c-c-c c-c c-c-c-c-c-c CO2 CO2 Oxalacetate c-c-c-c Citric Acid Cycle Glucose pyruvate c-c-c-c-c-c c-c-c pyruvate Acetyl CoA citric acid c-c-c c-c c-c-c-c-c-c CO2 CO2 Oxalacetate c-c-c-c Citric Acid Cycle Glucose pyruvate c-c-c-c-c-c c-c-c pyruvate Acetyl CoA citric acid c-c-c c-c c-c-c-c-c-c CO2 CO2 Oxalacetate c-c-c-c Citric Acid Cycle Glucose pyruvate c-c-c-c-c-c c-c-c pyruvate Acetyl CoA citric acid c-c-c c-c c-c-c-c-c-c CO2 CO2 Oxalacetate c-c-c-c  The Citric Acid Cycle Consists of Two Stages In the first stage of the citric acid cycle, two carbons are introduced into the cycle by condensation of an acetyl group with a four- carbon compound, oxaloacetate. The six-carbon compound formed (citrate) undergoes two oxidative decarboxylations, generating two molecules of CO2. In the second stage, oxaloacetate is regenerated. Both stages generate high-energy electrons that are used to power the synthesis of ATP in oxidative phosphorylation. Diagram of Cellular Respiration  condensation Citric Acid Cycle Step 1 isomerization Step 2 oxidation oxidation decarboxylation Step 8 Step 3 hydration oxidation Step 7 decarboxylation Step 4 Step 6 hydrolysis oxidation Step 5  Reaction 1: Formation of Citrate In the first reaction of the citric acid cycle, citrate synthase catalyzes the condensation of an acetyl group (2C) from acetyl CoA with oxaloacetate (4C) to yield citrate (6C) and coenzyme A. the energy to form citrate is provided by the hydrolysis of the high-energy thioester bond in acetyl CoA. Two Step Reaction to Form Citrate  Structures of The Conformational Changes in Citrate Synthase on Binding Oxaloacetate Citrate synthase exhibits induced fit. Oxaloacetate binding by citrate synthase induces structural changes that lead to the formation of the acetyl CoA binding site. The formation of the reaction intermediate citryl CoA causes a structural change that completes active site formation. Citryl CoA is cleaved to form citrate and coenzyme A.  Reaction 2: Isomerization In reaction 2 of the citric acid cycle, citrate rearranges to isocitrate, a secondary alcohol. aconitase catalyzes the dehydration of citrate (tertiary alcohol) to yield cis-aconitate, followed by a hydration that forms isocitrate (secondary alcohol).  Reaction 3: Oxidation, Decarboxylation In reaction 3, isocitrate undergoes decarboxylation by isocitrate dehydrogenase. One carbon is removed by converting a carboxylate group (COO−) to CO2. The dehydrogenase removes hydrogen ions and electrons, used to reduce NAD+ to NADH and H+.  Isocitrate Is Oxidized and Decarboxylated to Alpha- Ketoglutarate Isocitrate dehydrogenase catalyzes the oxidative decarboxylation of isocitrate, forming α-ketoglutarate and capturing high-energy electrons as NADH.  Reaction 4: Decarboxylation, Oxidation In reaction 4, catalyzed by α-ketoglutarate dehydrogenase, α-ketoglutarate (5C) undergoes decarboxylation to yield (4C) succinyl CoA. oxidation of the thiol group (— SH) in HS — CoA provides hydrogen that is transferred to NAD+ to form a second molecule of NADH and H+.  The enzyme and the reactions are structurally and mechanistically similar to the pyruvate dehydrogenase complex.  condensation Citric Acid Cycle Step 1 isomerization Step 2 oxidation oxidation decarboxylation Step 8 Step 3 hydration oxidation Step 7 decarboxylation Step 4 Step 6 hydrolysis oxidation Step 5  Reaction 5: Hydrolysis In reaction 5, catalyzed by succinyl CoA synthetase, hydrolysis of the thioester bond in succinyl CoA yields succinate and HS — CoA. energy from hydrolysis is transferred to the condensation of phosphate and GDP forming GTP, a high-energy compound similar to ATP. ADP + Pi ATP or or GTP = anabolic reactions (liver) ATP = cellular respiration (skeletal/heart muscle)  Reaction Mechanism of Succinyl CoA Synthetase Cleavage of the thioester of succinyl CoA powers the formation of ATP. The formation of ATP by succinyl coenzyme A synthetase is an example of a substrate-level phosphorylation because succinyl phosphate, a high- phosphoryl-transfer-potential compound, donates a phosphate to ADP.  Reaction 6: Oxidation In reaction 6, catalyzed by succinate dehydrogenase, succinate is oxidized to fumarate, a compound with a C = C bond. 2H lost from succinate are used to reduce the coenzyme FAD to FADH2.  Reaction 7: Hydration In reaction 7, catalyzed by fumarase, water is added to the double bond of fumarate to yield malate, a secondary alcohol.  Reaction 8: Oxidation In reaction 8, catalyzed by malate dehydrogenase, the hydroxyl group in malate is oxidized to a carbonyl group, yielding oxaloacetate. oxidation provides hydrogen ions and electrons for the reduction of NAD+ to NADH and H+.  condensation Citric Acid Cycle Step 1 isomerization Step 2 oxidation oxidation decarboxylation Step 8 Step 3 hydration oxidation Step 7 decarboxylation Step 4 Step 6 hydrolysis oxidation Step 5  Summary, Citric Acid Cycle In the citric acid cycle, an acetyl group bonds with oxaloacetate to form citrate. two decarboxylations remove two carbons as two CO2. four oxidations provide hydrogen for three NADH and one FADH2. a direct phosphorylation forms GTP (ATP).  The Citric Acid Cycle Produces High-Transfer-Potential Electrons, an ATP, and Carbon Dioxide The net reaction of the citric acid cycle is The electrons from NADH will generate 2.5 ATP when used to reduce oxygen in the electron-transport chain. The electrons from FADH2 will power the synthesis of 1.5 ATP with the reduction of oxygen in the electron-transport chain.  The Citric Acid Cycle Is Regulated The citric acid cycle is controlled at several points. The key control points in the citric acid cycle are the reactions catalyzed by isocitrate dehydrogenase and α-ketoglutarate dehydrogenase. Recall that pyruvate dehydrogenase controls entry of glucose-derived acetyl CoA into the cycle.  The Citric Acid Cycle Is a Source of Biosynthetic Precursors Many of the components of the citric acid cycle are precursors for biosynthesis of key biomolecules.  The Citric Acid Cycle Must Be Capable of Being Rapidly Replenished Because the citric acid cycle provides precursors for biosynthesis, reactions to replenish the cycle components are required if the energy status of the cells changes.  The Citric Acid Cycle Must Be Capable of Being Rapidly Replenished These replenishing reactions are called anaplerotic reactions. A prominent anaplerotic reaction is catalyzed by pyruvate carboxylase, which synthesizes oxaloacetate by the carboxylation of pyruvate. Recall that this reaction is also used in gluconeogenesis and is dependent on the presence of acetyl CoA.  Question 1 Malonate is a competitive inhibitor of succinate dehydrogenase. How will the concentrations of citric acid cycle intermediates change immediately after the addition of malonate? Why is malonate not a substrate for succinate dehydrogenase?  Study Check How many of each of the following are produced in one turn of the citric acid cycle? A. _ CO2 B. _ NADH C. _ FADH2 D. _ GTP  Solution How many of each of the following are produced in one turn of the citric acid cycle? A. 2 CO2 B. 3 NADH C. 1 FADH2 D. 1 GTP  Question 2 Which enzyme of the citric acid cycle most closely resembles the pyruvate dehydrogenase complex in terms of its structure, organization, and the reaction it performs? A. isocitrate dehydrogenase B. α-ketoglutarate dehydrogenase C. succinate dehydrogenase D. malate dehydrogenase  Question 3 As the citric acid cycle proceeds from the entry of acetyl CoA to the production of succinate, two carbons enter the cycle and two carbons are released as CO2. Why is the cycle not considered complete at this point? A. Not enough NADH has been generated from the cycle at this stage. B. The cell requires FADH2, which is produced by subsequent reactions of the cycle. C. The oxaloacetate used to initiate the cycle must be regenerated. D. Not enough energy has been generated from the cycle at this stage.  Question 4 An experiment labels pyruvate with radioactive C at C-2, which is the middle keto carbon. Where would this radiolabel appear after one turn of the citric acid cycle? A. as CO2 B. in one carboxyl group of oxaloacetate C. equally divided between the two carboxyl groups of oxaloacetate D. on the methylene carbon, −CH2, of oxaloacetate  Question 5 In the reaction catalyzed by succinyl CoA synthetase, A. a phosphohistidine residue is used to displace conenyzme A from succinate. B. a succinyl phosphate transfers its phosphate to a histidine residue. C. GDP is phosphorylated by a succinyl phosphate. D. GTP is the only high-energy compound produced.  Question 6 Why is acetyl CoA an especially appropriate activator for pyruvate carboxylase?  Question 7 PROBLEM: Fluoroacetate is a toxic molecule that inhibits the citric acid cycle. When fluoroacetate is added to mitochondria, fluorocitrate builds up. a) What step in the citric acid cycle is inhibited by fluoroacetate treatment? b) What would be the effect of the inhibition on other intermediates in the citric acid cycle?  Problem-Solving Strategies SOLUTION: Let’s again break down this question into a series of smaller questions, beginning with a simple but crucial question. Fluoroacetate is an analog of what molecule? If we look at the structure of fluoroacetate, it looks like an acetate with fluorine replacing a hydrogen. The fact that fluorocitrate is formed tells us something about the immediate metabolic fate of fluoroacetate. As a hint, think about how acetyl groups enter the citric acid cycle.  Problem-Solving Strategies SOLUTION: How is it possible for fluoroacetate to enter the citric acid cycle? It must first be converted to fluoroacetyl CoA. Good. How do you think fluorocitrate can be formed? Fluoroacetyl CoA must react with oxaloacetate to form fluorocitrate. Thinking back to the citric acid cycle: What is the normal fate of citrate formed by the condensation of acetyl CoA with oxaloacetate? Citrate is converted into isocitrate by the enzyme aconitase. Now to answer part (a), given what we just remembered: What step in the citric acid cycle is inhibited by fluorocitrate?  Problem-Solving Strategies SOLUTION: The aconitase reaction. It appears the fluorocitrate is not a substrate for aconitase, so it accumulates. Good job. On to part (b). If the cycle is blocked at the aconitase reaction: What would be the effect on the reactions following the aconitase-catalyzed step? All of the reactions would continue, beginning with the processing of all of the isocitrate formed before the introduction of the fluoroacetate. However, no new isocitrate could be generated because of the inhibition of aconitase.  Problem-Solving Strategies SOLUTION: What citric acid cycle intermediate would be regenerated? Oxaloacetate What would be the fate of oxaloacetate if sufficient fluoroacetate were present? It would react with fluoroacetyl CoA until all citric acid cycle components were converted into fluorocitrate. The citric acid cycle would halt, a situation most definitely not compatible with life.  Question 8 The pyruvate dehydrogenase complex is subject to allosteric control, especially inhibition by reaction products. The main regulatory process controlling pyruvate dehydrogenase's activity in eukaryotes is A. phosphorylation by ATP, which turns the complex off, and dephosphorylation, which turns the complex on. B. phosphorylation by ATP, which turns the complex on, and dephosphorylation, which turns the complex off. C. exchange of ADP and ATP on the pyruvate dehydrogenase complex. D. AMP binding to and activating the enzyme.  Question 9 Anaplerotic reactions serve to A. divert materials out of the citric acid cycle for use in biosynthesis. B. produce pyruvate, which initiates the cycle when glucose degradation is not occurring. C. replenish the citric acid cycle if it becomes depleted of intermediates by biosynthetic demands. D. regulate energy production by bypassing the citric acid cycle.  Question 10 Although the glyoxylate cycle is not present in animals, many plants and microorganisms use this modified form of the citric acid cycle. The glyoxylate cycle is beneficial in that A. it enables these organisms to grow on acetate. B. provides a source of glyoxylate, which is an essential biosynthetic intermediate in these organisms. C. does not release CO2, which is toxic to these organisms. D. is an alternative pathway for the generation of electron carriers in the absence of oxygen.