BCH 3303 Biochemistry Overview

BCH 3303 Essentials of Biochemistry

• This is week 10, lecture 19, 3rd April 2025.
• The class runs Tuesdays and Thursdays from 10:00 - 11:15AM in McKinney Humanities 2.01.44
• The instructor is Syed Muhammad Usama, PhD, Assistant Professor, Department of Chemistry.
• Email is [email protected], office phone is 210-458-5641
• Office hours are Mondays and Wednesdays 10:00 - 12:00 PM in office BSE 1.104C or by appointment.
• The grader is Thomas Yost, Biochemistry Major, 2025, [email protected]
• Homework Assignment (Chapter 16 and 17) is due on Friday, April 4th at 11:59 PM
• Homework Assignment (Chapter 18 and 19) is due on Friday, April 11th at 11:59 PM
• The required material is Biochemistry: A Short Course Fourth Edition by John L. Tymoczko, Jeremy M. Berg, Gregory J. Gatto, Jr., and Lubert Stryer

Semester Schedule

• Week 1: Chapter 1
• Week 2: Chapter 2, 3
• Week 3: Chapter 4, 5
• Week 4: Chapter 6, 7; Exam 1; Module 1-3; Thursday, 13th February
• Week 5: Chapter 7, 8
• Week 6: Chapter 9, 10
• Week 7: Chapter 11, 14; Exam 2; Module 4-6; Thursday, 6th March
• Week 8: Chapter 15, 16
• Week 9: Chapter 16, 17
• Week 10: Chapter 18, 19; Exam 3; Module 7-9; Thursday, 3rd April
• Week 11: Chapter 20, 26
• Week 12: Chapter 27, 33
• Week 13: Chapter 34, 39; Exam 4; Module 10 - 12; Thursday, 24th April
• Week 14: Chapter 40; Final Exam; Module 1-14; Tuesday, 13th May

The Synthesis of Acetyl Coenzyme A from Pyruvate

• Acetyl CoA synthesis from pyruvate proceeds in three steps: decarboxylation, oxidation, and CoA transfer

Pyruvate Dehydrogenase Complex of E. coli

• Pyruvate dehydrogenase complex requires a prosthetic group that is essential for enzyme activity and covalently attached
• Stoichiometric coenzymes like CoA and NAD+ function as substrates
• Catalytic coenzymes like TPP, lipoic acid, and FAD are not permanently altered during the reaction

Decarboxylation

• Pyruvate dehydrogenase (E₁) catalyzes decarboxylation
• Pyruvate combines with the ionized coenzyme form, thiamine pyrophosphate (TPP)

Oxidation

• The hydroxyethyl group attached to TPP oxidizes to form an acetyl group while being simultaneously transferred to lipoamide
• Lipoamide, a derivative of lipoic acid, has its disulfide group reduced to a disulfhydryl form
• E₁ catalyzes the reaction, yielding acetyl-lipoamide

Formation of Acetyl CoA

• The transfer of the acetyl group from acetyl-lipoamide to coenzyme A to form acetyl CoA is catalyzed by E2

The regeneration of Dihydrolipoamide

• Dihydrolipoamide must be reoxidized to participate in another reaction cycle
• Dihydrolipoamide dehydrogenase (E3) catalyzes this reaction

Regulation of the Pyruvate Dehydrogenase

• The formation of acetyl CoA from pyruvate is irreversible in animal cells
• Acetyl CoA has two principle fates: metabolism via the citric acid cycle or incorporation into fatty acids

Phosphorylation/Dephosphorylation of Enzyme E₁

• Enzyme E₁ is a key site of regulation

Energy Charge

• Pyruvate dehydrogenase complex is also regulated by energy charge
• ATP, acetyl CoA, and NADH inhibit the complex
• ADP and pyruvate stimulate the complex

Harvesting Electrons from the Cycle

• The figure depicts Acetyl CoA turning into 2CO2, with ATP and 8e- as products.

Citric Acid Cycle

• Glucose turns into pyruvate, and pyruvate yields acetyl CoA
• Acetyl CoA and oxaloacetate react to produce citric acid.

The Citric Acid Cycle Consists of Two Stages

• Stage 1: introduction of two carbons into the cycle via condensation of an acetyl group with oxaloacetate, a four-carbon compound
• The six-carbon compound formed (citrate) undergoes two oxidative decarboxylations, generating two molecules of CO2
• Stage 2: oxaloacetate is regenerated
• Both stages generate high-energy electrons that power ATP synthesis in oxidative phosphorylation

Diagram of Cellular Respiration

• Fatty acids, glucose, and amino acids enter the citric acid cycle.
• Acetyl CoA yields 1 ATP and 2CO₂
• An electron-transport chain, with NADH and FADH₂, yields 2O₂ and 4H₂O
• A proton gradient gives ATP
• A Matrix contains the inner mitochondrial membrane

Citric Acid Cycle Steps

• Step 1: Condensation
• Step 2: Isomerization
• Step 3: oxidation decarboxylation
• Step 4: oxidation decarboxylation
• Step 5: hydrolysis
• Step 6: oxidation
• Step 7: hydration
• Step 8: oxidation

Reaction 1: Formation of Citrate

• Citrate synthase catalyzes the condensation of an acetyl group (2C) from acetyl CoA with oxaloacetate (4C) to yield citrate (6C) and coenzyme A
• Energy to form citrate stems from the hydrolysis of the high-energy thioester bond in acetyl CoA

Structures of The Conformational Changes in Citrate Synthase on Binding Oxaloacetate

• Citrate synthase exhibits induced fit
• Oxaloacetate binding by citrate synthase causes structural changes that lead to the formation of the acetyl CoA binding site
• The reaction intermediate citryl CoA formation causes a structural change that completes active site formation
• Citryl CoA is cleaved to form citrate and coenzyme A

Reaction 2: Isomerization

• 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

• 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+

Reaction 4: Decarboxylation, Oxidation

• 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+.

Reaction 5: Hydrolysis

• Catalyzed by succinyl CoA synthetase, thioester bond hydrolysis in succinyl CoA yields succinate and HS — CoA
• Energy from hydrolysis is transferred to phosphate/GDP condensation, forming GTP, a high-energy compound like ATP

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 a substrate-level phosphorylation example.
• Succinyl phosphate, a high-phosphoryl-transfer-potential compound, donates a phosphate to ADP.

Reaction 6: Oxidation

• Succinate is oxidized to fumarate, a compound with a C = C bond by succinate dehydrogenase
• 2H lost from succinate are used to reduce the coenzyme FAD to FADH2

Reaction 7: Hydration

• Water is added to the double bond of fumarate to yield malate, a secondary alcohol via fumarase

Reaction 8: Oxidation

• The hydroxyl group in malate is oxidized to a carbonyl group, yielding oxaloacetate by malate dehydrogenase
• Oxidation provides hydrogen ions and electrons for reduction of NAD+ to NADH and H+.

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 Summary Yields

• 2 CO2
• 3 NADH and 3H+
• 1 FADH2
• 1 GTP (1 ATP)
• 1 HS—CoA

The Citric Acid Cycle Produces High-Transfer-Potential Electrons, an ATP, and Carbon Dioxide

• The net reaction of the citric acid cycle is Acetyl CoA + 3 NAD+ + FAD + ADP + P₁ + 2 H2O -> 2 CO₂ + 3 NADH + FADH2 + ATP + 2 H+ + CoA
• 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 are the reactions catalyzed by isocitrate dehydrogenase and α-ketoglutarate dehydrogenase
• Pyruvate dehydrogenase controls entry of glucose-derived acetyl CoA into the cycle

The Citric Acid Cycle Is a Source of Biosynthetic Precursors

• Cycle components are precursors for biosynthesis of key biomolecules

The Citric Acid Cycle Must Be Capable of Being Rapidly Replenished

• reactions to replenish cycle components are required if energy status of the cells changes

Anaplerotic Reactions

• Replenishing reactions that are catalyzed by pyruvate carboxylase, which synthesizes oxaloacetate, dependent on the presence of acetyl CoA
• Reaction Pyruvate + CO2 + ATP + H₂O -> oxaloacetate + ADP + P₁ + 2 H+

Question 1

• Malonate is a competitive inhibitor of succinate dehydrogenase
• Succinate will increase in concentration, followed by α-ketoglutarate and the other intermediates "upstream" of the site of inhibition.
• Succinate has two methylene groups required for dehydrogenation, whereas malonate has only one

Study Check

• One turn of the citric acid cycle produces:
  • 2 CO2
  • 3 NADH
  • 1 FADH2
  • 1 GTP

Question 2

• α-ketoglutarate dehydrogenase most closely resembles the pyruvate dehydrogenase complex in terms of its structure, organization, and the reaction it performs

Question 3

• The oxaloacetate used to initiate the cycle must be regenerated

Question 4

• pyruvate is labeled with radioactive C at C-2, equally divided between the two carboxyl groups of oxaloacetate

Question 5

• Succinyl phosphate transfers its phosphate to a histidine residue

Question 6

• Pyruvate carboxylase should be active only when the acetyl CoA concentration is high
• Acetyl CoA might accumulate if energy needs aren't being met because of an oxaloacetate deficiency
• Alternatively, acetyl CoA might accumulate because energy needs of the cell have been met.
• Pyruvate will be converted back into glucose, and the first step in this conversion is the formation of oxaloacetate

Question 7

• Fluoroacetate is a toxic molecule that inhibits the citric acid cycle where fluorocitrate builds up, that is in the aconitase reaction

Question 8

• phosphorylation by ATP, which turns the complex off, and dephosphorylation, which turns the complex on, is the primary method of regulation of pyruvate dehydrogenase's activity in eukaryotes

Question 9

• Anaplerotic reactions replenish the citric acid cycle if it becomes depleted of intermediates by biosynthetic demands

Question 10

• Glyoxylate cycle enables organisms to grow on acetate