Exam 2 - Cell Bio

Introduction to Enzymes

Enzymes are specialized proteins that function as biological catalysts, lowering the activation energy required for chemical reactions, thus facilitating and speeding up these processes. They play a vital role in regulating biochemical pathways within cells, influencing various metabolic reactions essential for life.

Mechanisms by which Enzymes Lower Activation Energy

• Aligning Reactants: Enzymes precisely position substrates closer together within their active site, enhancing the likelihood of effective collisions and interactions. This spatial arrangement increases the probability of reaction due to improved orientation compared to unassisted reactions.

• Changing Reactivity: Enzymes can modify the chemical properties of substrates through electrostatic interactions. The electrical charges present on the amino acids that comprise the enzymes can stabilize the transition state of the reaction, thereby facilitating the formation of products while reducing the energy barrier.

• Stress Methodology: The binding of a substrate to an enzyme results in conformational changes to the enzyme's structure, creating physical stress on the substrate’s chemical bonds. This tension can lead to the breaking or rearranging of bonds, making it easier for the reaction to occur. An example is helicase, which unwinds DNA by exerting stress on the hydrogen bonds that hold the double helix together.

Enzymatic Structures and Cofactors

Enzymes often require the assistance of additional non-protein molecules to achieve optimal functionality:

• Apoenzyme: This is the inactive form of an enzyme that requires specific cofactors to become active.

• Cofactors: These are necessary nonprotein molecules, often metal ions (like zinc or magnesium) or organic compounds (like vitamins), that enhance enzymatic activity.

• Holoenzyme: This term refers to the active form of an enzyme, which includes both the apoenzyme and its associated cofactors.

The Lock and Key Model

The interaction between enzymes and their substrates can be likened to a lock and key. The enzyme’s active site is specifically shaped to fit the substrate, which ensures a high degree of specificity in enzyme-substrate interactions, essential for proper metabolic function.

ATP: Energy Currency of the Cell

• Adenosine Triphosphate (ATP): ATP is a ribonucleotide containing three phosphate groups, and it is the primary energy carrier in cells. The high-energy bonds between these phosphate groups store energy, which can be released for cellular processes when converted to adenosine diphosphate (ADP) through hydrolysis.

Metabolism Overview

• Catabolism: This is the metabolic pathway that breaks down macromolecules to release energy, such as during glycolysis, which involves the breakdown of glucose.

• Anabolism: This is the pathway that synthesizes macromolecules, requiring energy input facilitated by ATP.

Glycolysis Overview

Glycolysis is a ten-step metabolic pathway that breaks down glucose, a six-carbon sugar, into two three-carbon molecules of pyruvate:

• Stage 1: The investment phase where glucose is phosphorylated to form glucose 6-phosphate, catalyzed by hexokinase, utilizing one molecule of ATP.

• Stage 2: The energy generation phase involves the conversion of intermediate products into pyruvate, yielding a net gain of ATP and NADH.

Key Enzymes in Glycolysis

• Hexokinase: The first enzyme in the glycolysis pathway that phosphorylates glucose, thereby facilitating its entry into the metabolic pathway.

• Phosphofructokinase (PFK): This enzyme acts as a major regulatory point in glycolysis; it determines the pathway's flux and is sensitive to the energy state of the cell.

• Pyruvate Kinase: It catalyzes the final step of glycolysis, converting phosphoenolpyruvate to pyruvate and producing ATP in the process.

Enzyme Regulation in Glycolysis

The activity of glycolytic enzymes is closely regulated based on the cell's energy needs:

• High concentrations of ATP inhibit key glycolytic enzymes, ensuring that the pathway is downregulated when energy levels are sufficient.

• Hormones such as insulin and glucagon play crucial roles in regulating blood glucose levels, promoting or inhibiting glycolysis and influencing glycogen storage.

Delta G and Enzyme Catalysis

• Delta G: This term describes the change in free energy associated with a reaction.

  • A negative Delta G indicates that a reaction can proceed spontaneously, while a positive Delta G signifies that the reaction requires energy input (non-favorable).

  • Delta G is critical in understanding enzyme activity and the overall direction of metabolic pathways, guiding the design of metabolic interventions.

Glycogen Metabolism

Glycogen serves as a rapid and extensive source of glucose when the body requires energy.

• The liver plays a key role in storing glycogen and releasing glucose into the bloodstream as necessary, helping regulate blood sugar levels and energy availability.

• Cyclic AMP (cAMP) acts as a secondary messenger within cells, indicating the cellular energy status and regulating various metabolic processes.

Conclusion

The intricate regulation of enzymes through mechanisms such as phosphorylation and dephosphorylation by kinases and phosphatases is paramount for metabolic homeostasis, influencing energy production and storage dynamics within cells. Understanding these enzymatic functions and regulations is fundamental for grasping metabolic pathways and their implications in health and disease.

Overview of Metabolism

• Anabolism and Catabolism

  • Anabolism is the first metabolic process, involved in building complex molecules from simpler ones.

  • Not all cells participate equally in metabolic processes (e.g., anabolism, catabolism).

Historical Context

• Carl and Gertie Cori

  • Won the Nobel Prize in 1947 for their research on metabolism.

  • Focused on how the body processes substances during metabolism, specifically glucose.

Types of Cells in Metabolism

• Muscle Cells vs. Liver Cells

  • Muscle cells have limited aerobic capacity which leads to lactate production under strenuous activity.

  • Lactate travels through blood back to liver cells for further processing.

  • Liver cells are more proficient in gluconeogenesis compared to muscle cells.

Glycolysis and Gluconeogenesis

• Glycolysis

  • Key pathway that converts glucose into pyruvate.

  • Certain steps are easily reversible, primarily characterized by neutral delta G values.

  • Specific enzymes, such as isomerases and kinases, play important roles in this regulation.

• Gluconeogenesis

  • Primarily occurs in the liver; muscle cells cannot undergo this process adequately due to different enzyme availability.

  • Regulatory sites for this process are influenced by the availability of ATP and other nucleotide triphosphates like GTP.

Regulation of Metabolic Pathways

• Hormonal Regulation

  • The hypothalamus regulates gluconeogenesis via chemical messengers through the bloodstream.

  • Metformin, used in diabetes treatment, inhibits gluconeogenesis, reflecting the importance of regulation in metabolism.

• Feedback Mechanisms

  • Feedback inhibition occurs when high levels of ATP signal a slowdown in glycolysis, affecting enzymes like phosphofructokinase.

  • Conversely, an accumulation of fructose 1,6-bisphosphate stimulates downstream enzymes like pyruvate kinase.

Fermentation and Anaerobic Processes

• Response to Low Oxygen

  • Under anaerobic conditions, pyruvate may undergo fermentation processes, leading to the production of ethanol and carbon dioxide.

  • The body utilizes mechanisms to recycle NAD+ to ensure glycolysis can continue even during low oxygen availability.

Acetyl CoA and Citric Acid Cycle

• Acetyl CoA Formation

  • Pyruvate can be converted into Acetyl CoA, which enters the citric acid cycle (Krebs Cycle) for further energy production.

  • This is a key intersection in metabolic pathways, showcasing versatility in energy usage.

Membrane Structure and Function

• Cell Membrane Composition

  • Biological membranes are primarily made of a phospholipid bilayer, consisting of two layers of lipids (leaflets).

  • The polar heads face the aqueous environment, while fatty acid tails point inward.

  • Membranes also include protein channels for the transportation of polar molecules.

• Fluid Mosaic Model

  • Proposed by Singer and Nicholson; describes the dynamic and flexible nature of the cell membrane structure, which is continuously maintained.

  • The model explains the distribution and organization of proteins within the lipid bilayer.

Phospholipids and Membrane Structure

• Basic Structure of Phospholipids

  • Composed of fatty acid tails that can be saturated or unsaturated.

  • Contains glycerol backbone.

  • Various head groups exist, including simple phosphate (e.g., phosphatidic acid) to complex structures like diphosphatidylglycerol.

  • Diversity of phospholipids contributes to the complexity of biological membranes.

Fatty Acid Tails

• Role of Fatty Acids

  • Nonpolar tails orient towards the interior of the membrane.

  • Saturated fatty acids pack tightly, leading to a more solid membrane.

  • Unsaturated fatty acids have bent tails, preventing tight packing, resulting in a liquid-like membrane.

  • The fluidity of the membrane is determined by the saturation of fatty acids, influencing membrane function.

Cholesterol in Membranes

• Function of Cholesterol

  • Features a polar head and nonpolar steroid rings; acts as a membrane stabilizer.

  • Helps maintain membrane integrity by filling gaps left by unsaturated fatty acids, preventing water and other substances from disrupting the bilayer.

  • Acts as a temperature buffer: stabilizes membrane at high temperatures and maintains fluidity at low temperatures.

Membrane Composition and Function

• Types of Lipids

  • Fatty acids: Primary structure components of phospholipids.

  • Glycolipids: Include sugars, affecting cell signaling and interactions; removal can lead to diseases.

  • Phospholipid Variability: Different cell types have distinct lipid compositions, which are adapted to their specific functions (e.g., red blood cells vs. brain cells).

Asymmetrical Leaflets

• Two sides of the lipid bilayer (outer and inner) differ compositionally to fulfill varying functions.

• Outer leaflet engages with the external environment; inner leaflet is involved in intracellular signaling.

Transmembrane Proteins

• Types of Membrane Proteins

  • Integral Proteins: Span the membrane; significant role in transport and communication.

  • Peripheral Proteins: Attached to the membrane’s surface; do not penetrate the bilayer.

  • Lipid-Linked Proteins: Covalently bonded to lipid tails, influencing membrane dynamics and interaction.

Predicting Membrane Proteins

• Identifying Transmembrane Domains

  • Software such as HypeDoolittle and Transmembrane Finder can analyze protein sequences to predict transmembrane domains using hydrophobicity profiles.

  • Beta Barrel Proteins: Found in prokaryotes and mitochondria; allow passive transport across membranes.

Cell Membrane Fluidity and Dynamics

• Membrane composition affects fluidity; saturated lipids contribute to solid-like characteristics, while unsaturated lipids enhance fluidity.

• Membrane Movement:

  • Lateral movement of phospholipids is common; flipping across the bilayer is rarer due to energetical constraints.

  • Techniques to study membrane dynamics include fluorescence recovery after photobleaching (FRAP) to measure mobility and fluidity of membrane components.

Lipid Rafts

• Cholesterol and sphingolipid-rich domains exhibit unique properties; these lipid rafts facilitate protein movement and cellular signaling.

• Areas with different lipid compositions can impact the behavior of proteins, influencing the overall cell response.

Conclusion

• Membrane architecture is essential for cell function; involves a variety of lipids, proteins, and their specific arrangements.

• Understanding membrane composition and dynamics is crucial for insight into cellular processes and potential therapeutic targets.

Introduction to the Active Cytoskeleton

• The active cytoskeleton plays a crucial role in the organization and mobility of proteins within the cell.

• Proteins are situated in a liquid crystal membrane and are influenced by an underlying actin lattice.

Actin Filaments and Protein Mobility

• The actin filament lattice acts like fences, impeding protein movement within the membrane.

• This concept is illustrated by comparing protein movement to navigating through subdivision fences in Texas.

• Hypothesis: Cellular structures (fences) localize proteins to specific cell regions, such as neurons.

The Piggy Fence Model

• A model proposed in the early 2000s explaining protein localization in neurons.

• Neurons have specific proteins located in the cell body and axon, with mechanisms preventing random movement.

• Fences formed by actin and binding proteins help to maintain the organization of proteins within the cell.

Actin Binding Proteins and Protein Identification

• Actin-binding proteins are crucial in forming the cytoskeletal meshwork.

• Studies on red blood cells highlighted the relationship between these proteins and their structural integrity:

  • Band 3, Band 4.1, Band 4.2 were identified using SDS-PAGE to determine their interactions with the actin cytoskeleton.

• Spectrin locks actin filaments together, forming a supportive network for membrane proteins.

Importance of Structural Integrity

• Actin and spectrin interact to maintain the red blood cell's characteristic disc shape.

• Loss of spectrin leads to failure in maintaining cell structure, resulting in premature cell death through filtering by the liver.

• Can lead to hemolytic anemia, where lack of red blood cells impairs oxygen delivery.

Evolution of Protein Domains

• Protein domains linked to actin and spectrin are crucial for various cellular functions.

• Domains allow proteins to interact at specific locations, essential for neuron function (action potential) and muscle integrity.

• Dystrophin and dystroglycan are involved in muscle cell stability and are associated with muscular dystrophy when mutated.

Cell Structure Organization

• Different cell types demonstrate varied organization:

  • Intestinal Cells: Importance of protein location for nutrient transport (apical and basolateral surfaces).

  • Neurons: Organization of input and output proteins ensures effective signaling.

Membrane Fluidity and Regulation

• Fluidity is affected by saturated vs. unsaturated fatty acids and cholesterol presence.

• The active cytoskeleton influences the interactions between lipids and proteins, maintaining cell asymmetry.

• Membrane integrity is critical for cell function and organization, demonstrated through membrane studies.

Membrane Permeability and Transport Mechanisms

• Semipermeability of membranes implies selective transport of substances.

• Diffusion: Movement from high to low concentration is characterized without membrane interaction; can drive molecule movement across membranes.

• Transport Types: Includes non-mediated passive transport, ion channel-mediated transport, passive transporter-mediated transport, and active transport against gradients.

Osmosis Explained

• Osmosis: The specific movement of water across a semipermeable membrane from lower to higher solute concentrations.

• Osmosis is contrasted with diffusion where solute particles move in opposite directions.

• Practical examples include experiments with eggs in vinegar and engineering isotonic solutions for blood cell health.

Practical Implications of Membrane Transport

• Understanding osmotic balance is vital for medical applications, such as administering isotonic saline for patient care.

• Plants utilize osmotic movement for functions such as nutrient uptake (turgidity) and responses to touch (movement).

• Plasmolysis occurs when plant cells are placed in hypertonic solutions, leading to tissue damage.

Conclusion

• A robust understanding of how the active cytoskeleton aids in protein localization, membrane fluidity, and transport mechanisms is crucial for cellular biology studies.