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MCB 150: The Molecular and Cellular Basis of Life

Lecture 11: Cellular Respiration

Announcements

• No lecture or student hours on Friday.

• Pre- and post-class questions remain the same.

• Content is derived from Fall 2024 lecture recordings.

• Review exam question times:

  • Today: 3:30-5:00 PM

  • Tuesday: 10:30-Noon

  • Thursday: 9:30-11:00 AM

Phase 2: Pyruvate Oxidation and Krebs Cycle

Net Results for Each Glucose from Glycolysis and Krebs Cycle:

• Glycolysis converts one molecule of glucose into two molecules of pyruvate. Each pyruvate can then enter the Krebs cycle following pyruvate oxidation.

• Throughout glycolysis, a net of 2 ATP are produced, along with 2 NADH.

• In the Krebs cycle, each turn creates 3 NADH, 1 FADH2, and 1 GTP, producing 2 GTP (which can be converted to ATP).

• Collectively, the theoretical yield from one glucose molecule during the entire cellular respiration process is up to 36 ATP.

Problems at the End of the Krebs Cycle

1. Insufficient Replacement of NAD+: The excessive accumulation of NADH can inhibit the Krebs cycle because NAD+ is required for the dehydrogenase enzymes within the cycle to function.

2. Presence of FADH2: Similar to NADH, FADH2 must be re-oxidized to contribute to the electron transport chain (ETC).

3. Energy carried by cofactors not yet converted to ATP: Although energy from substrates has been transformed into cofactors, this energy still needs to be transferred into ATP form.

Oxygen Dependency

• Aerobic Respiration: Requires oxygen for processes beyond the Krebs cycle, particularly for the electron transport chain where oxygen acts as the terminal electron acceptor.

• Krebs Cycle: While it functions in the mitochondrial matrix and does not directly require oxygen, it does rely on a functional ETC for NADH and FADH2 re-oxidation to continue.

Oxidative Phosphorylation and Electron Transport Chain

NADH Functionality:

• Electrons are transferred from NADH to the first complex of the electron transport chain located in the inner mitochondrial membrane.

• During this process, NADH is oxidized back to NAD+, which can re-enter glycolysis and the Krebs cycle.

Electron Transport:

1. Sequential Energy Release: Electrons move through a series of protein complexes, losing energy with each transfer, which is harnessed to pump protons (H+) across the inner mitochondrial membrane.

2. Proton Gradient Formation: This creates a pronounced electrochemical gradient across the membrane, known as the proton motive force, which is essential for ATP synthesis during chemiosmosis.

3. Final Electron Transfer: Electrons are ultimately transferred to oxygen, producing water. This step is crucial for preventing an electron backlog in the chain and for accommodating the CO2 produced from the Krebs cycle and water from the ETC.

FADH2 Contribution:

• FADH2 enters the ETC at a lower energy level and contributes less to the proton gradient compared to NADH: creating a net yield of 2 ATP per FADH2 rather than 3 ATP per NADH.

Results of Electron Transport Chain

• Although cofactors are regenerated and a proton gradient is established, no ATP is produced directly during the electron transport chain phase itself.

• Key locations involved in this process: Cytosol, Intermembrane Space, Mitochondrial Matrix.

Theoretical ATP Production from Glucose Oxidation

1. Glycolysis: Produces a net of

   • 2 ATP + 4 ATP from the reoxidation of 2 NADH (2 ATP generated per NADH) totaling 6 ATP.

2. Krebs Cycle Contributions:

   • From Krebs cycle: 2 GTP, which equals 2 ATP.

   • 8 NADH = 24 ATP (8 NADH x 3 ATP each).

   • 2 FADH2 = 4 ATP (2 FADH2 x 2 ATP each).

3. Total Theoretical ATP production per Glucose: 36 ATP.

Lecture 12: Fermentation and Regulation of Metabolism

Student Hours and Availability

• Student hours today: 4:00-5:30 PM; can discuss general performance and preparation, but specifics of exams are off-limits.

• No lecture on Friday; a lecture video will be available through Canvas for review.

Pathway of Carbon and Hydrogen in Glucose Oxidation

• Glycolysis: Converts glucose into 2 pyruvate molecules.

• Subsequent pyruvate oxidation and Krebs Cycle liberate carbon dioxide (CO2), which is expelled as a waste product.

• The electron transport chain (ETC) and chemiosmosis ultimately yield water from the addition of hydrogen to oxygen, completing the process.

Conditions Under Anaerobic Respiration

• In the absence of oxygen, cells can still utilize glycolysis, but they cannot go further into Krebs cycle or oxidative phosphorylation.

• In this scenario, ATP yields are restricted solely to glycolysis, with the potential output being 2 ATP per glucose.

• Fermentation: To prolong glycolysis, pyruvate is transformed into lactic acid or ethanol to regenerate NAD+, allowing glycolysis to continue, although no additional ATP is generated since glucose remains partially oxidized.

• Accumulation of Byproducts: Such as lactic acid in muscles (resulting in fatigue) or ethanol in yeast (which also influences alcohol fermentations).

Types of Fermentation

• Yeast Fermentation:

  • Process involves Pyruvate → Acetaldehyde → Ethanol.

  • Key enzymes: Pyruvate Decarboxylase and Alcohol Dehydrogenase, which catalyze the conversion while regenerating NAD+.

• Muscle Cell Fermentation:

  • The process converts Pyruvate into Lactic Acid (Lactate) through the enzyme Lactate Dehydrogenase, also regenerating NAD+.

Regulation of Metabolic Pathways

• Energy Efficiency: The regulation of metabolic pathways is vital for maintaining cellular homeostasis. It is primarily coordinated through the modulation of enzyme amounts and their activity.

Allosteric Regulators:

• Binding Characteristics: These molecules bind at sites other than the active site on enzymes.

  • Positive Regulators: Enhance enzyme activity by inducing a conformational change allowing for increased substrate binding.

  • Negative Regulators: Decrease enzyme activity by similarly altering the conformation and reducing substrate binding.

Example of Feedback Inhibition in Glycolysis

• Phosphofructokinase (PFK) Regulation:

  • Mechanism: If ATP levels are high, ATP binds to the regulatory site, resulting in decreased activity of PFK (Negative Regulation). Conversely, high levels of ADP/AMP can increase PFK activity (Positive Regulation), driving glycolysis when energy is needed.

Advanced Regulation Mechanisms

• Allosteric regulators possess a unique capability to influence multiple metabolic pathways simultaneously, allowing cells to efficiently respond to varying energy demands by upregulating one pathway while downregulating another.

Central Dogma of Molecular Biology

DNA Functionality:

• Genetic Blueprint: DNA contains the genetic instructions necessary for the synthesis of proteins and RNA, guiding cellular function and organism development. The flow of genetic information can be summarized as: DNA → RNA → Protein.

Discovering DNA Function

• Historical Context:

  • In the 1940s, scientists identified hereditary material within chromosomes made up of chromatin (DNA + protein). Initially, proteins were thought to be carriers of genetic information due to variability among amino acids, leading to misconceptions. The landmark discovery was the identification of DNA as the true genetic material.

Chargaff’s Rules and Discovering DNA Structure

• Chargaff’s Findings: He established base-pairing rules, indicating relationships such as A = T and C = G, foundational for understanding DNA’s double-helical structure.

Structural Discovery of DNA

• Rosalind Franklin & Maurice Wilkins: Utilized X-ray diffraction techniques to determine the helical nature of DNA.

• Watson & Crick's Contribution: They built upon previous findings to present a coherent model explaining the structure of DNA as a double helix with specific base-pairing.

Base Pairing in DNA

• Hydrogen Bonding: The complementary nature of purines (A and G) pairing with pyrimidines (C and T) stabilizes the double helical structure of DNA. Each base follows specific pairing rules, illustrated as: G pairs with C and A pairs with T (known as Watson-Crick base pairing).

Antiparallel & Complementary Structure of DNA

• The two strands of DNA are not only complementary but also oriented in opposite directions (antiparallel), which allows for the storage of genetic information in the precise order of nucleotides, enhancing genetic variability through combinations of bases.

DNA Replication Overview

• Key Steps in DNA Replication:

  1. Identify the origin of replication.

  2. Separation of DNA strands.

  3. Priming process involving RNA primers.

  4. Synthesis of new DNA strands.

  5. Cleanup of Okazaki fragments and finalizing DNA strands completion.

Challenge of Antiparallel Synthesis

• Since DNA synthesis can only occur in the 5' to 3' direction, it leads to a differentiated replication mode where a leading strand is synthesized continuously and a lagging strand is synthesized in short segments known as Okazaki fragments.

RNA Primers in DNA Synthesis

• Primase Role: RNA primers, synthesized by the enzyme Primase, provide the 3'-OH group necessary for DNA polymerases to initiate synthesis of the new DNA strands.

DNA Polymerase I Role

• Functionality: DNA Polymerase I is responsible for removing RNA primers and replacing them with DNA nucleotides through its 5'–3' exonuclease activity, facilitating a seamless transition to newly synthesized DNA.

Importance of DNA Ligase

• Sealing Nicks: DNA Ligase plays a critical role in concatenating and sealing nicks resulting from DNA synthesis or repair processes, ensuring the integrity and continuity of the DNA strands.

Proofreading Mechanisms

• Error Correction: DNA polymerases possess proofreading capabilities, employing exonuclease activity to rectify any misincorporated nucleotides, thereby enhancing the fidelity of DNA replication.

Chromosomal Organization in Bacteria and Eukaryotes

• Bacterial DNA: Typically exists in compact, supercoiled forms maintained by topoisomerases, allowing efficient storage and organization.

• Eukaryotic DNA: Organized into nucleosomes; these structures are further compacted into chromatin fibers aiding in DNA stability, expression, and replication.

Eukaryotic Gene Structure

• Significant differences in gene structures, with the presence of non-coding regions (introns/exons), necessitating mRNA processing after transcription for proper expression and translation into functional proteins.

Summarizing DNA Replication

• The DNA replication process is fundamentally semiconservative, whereby each new DNA double helix comprises one parental strand and one newly synthesized strand, necessitating coordinated action of numerous molecular players for successful replication.