Enzymes and Metabolism Study Notes

Introduction to Enzymes

• Role of Enzymes: Biological catalysts that speed up reactions without altering equilibrium, essential for sustaining life by facilitating critical cellular processes.
• Biochemical Reactions: Enzymes are critical in facilitating about 10,000 biochemical reactions in cells, influencing processes such as digestion, metabolism, and DNA replication.
• Nature of Enzymes: Mostly proteins, though some RNA (ribozymes) have enzymatic activity, highlighting the versatility of biological molecules.
• Catalytic Mechanism: Enzymes follow the substrate + enzyme → product + enzyme model; they are recycled after reactions, allowing them to participate in multiple catalytic cycles without being consumed.
• Enzyme Specificity: Enzymes are named with the "-ase" suffix (e.g., glucose oxidase). Some act on multiple substrates, while others are highly specific to ensure precise biochemical pathways are followed.
• Catalysis & Thermodynamics: Enzymes lower activation energy (Ea) without changing the Gibbs free energy (ΔG) or reaction direction; they stabilize transition states that otherwise would require more energy to form.

Enzyme Specificity

• Enzyme Function and Energy Diagrams: Enzymes stabilize transition states to reduce activation energy, depicted in energy diagrams where the height of the activation energy barrier determines the rate of the reaction.
• Thermodynamics of Enzyme Activity: Enzymes do not alter ΔG, allowing them to facilitate reactions while maintaining the thermodynamic favorability or unfavorability of the reactions under physiological conditions.
• Enzyme Structure: Active enzymes are called holoenzymes, consisting of an apoenzyme (protein) and cofactors; cofactors may be metal ions or organic molecules (coenzymes) that assist in enzymatic activity.
• Models for Enzyme Action:
  • Lock-and-Key Model: Rigid specificity where the substrate fits perfectly into the enzyme’s active site.
  • Induced-Fit Model: Suggests flexibility in enzyme-substrate interaction, where the enzyme changes shape upon substrate binding to optimize the fit.
• Temperature & pH Sensitivity: Enzymes work best at optimal conditions; extreme deviations can lead to denaturation, while extremophiles have adapted enzymes for extreme pH and temperatures, enabling life in harsh environments.

Michaelis-Menten Kinetics

• Key Concepts:
  • Factors Affecting Enzyme Activity: pH, temperature, and inhibitors/activators influence enzyme function, demonstrating the importance of environmental conditions on enzymatic reactions.
  • Reaction Orders:
  • Zero-order: Rate independent of substrate concentration, often seen at high substrate levels.
  • First-order: Rate directly proportional to substrate concentration, applicable at low substrate levels.
• Michaelis-Menten Equation: Describes how reaction rates vary with substrate concentration, where:
  • Vmax = Maximum reaction velocity at enzyme saturation, indicating the maximum rate of reaction achievable.
  • Km = Substrate concentration at half Vmax, indicating enzyme affinity; low Km reflects high affinity for substrate, while high Km indicates lower affinity, indicating the enzyme’s efficiency.

Enzyme Parameters and Measurement

• Specific Activity (SA): Activity per gram of enzyme allows for cross-enzyme comparison, providing insight into the efficacy of different enzymes under identical conditions.
• Turnover Number (Kcat): Number of substrate molecules converted per enzyme active site per second, a key indicator of enzyme activity in catalytic processes. Higher purity increases specific activity.
• Methods for Measuring Km and Vmax:
  • Lineweaver-Burk Plot: Converts Michaelis-Menten data into a linear format; however, may exaggerate errors when estimating Km and Vmax.
  • Eadie-Hofstee Plot: Preferred method that avoids error magnification; it plots the reaction velocity against the ratio of velocity and substrate concentration, allowing for more accurate data interpretation.

Enzyme Regulation & Inhibition

• Importance of Regulation: Controls metabolic pathways to prevent overactivity, maintaining balance and efficiency in metabolic processes.
• Types of Inhibition:
  • Competitive: Inhibitor competes for active site; increases Km but not Vmax, effectively reducing substrate's apparent affinity.
  • Uncompetitive: Binds to ES complex; decreases both Km and Vmax, inhibiting the reaction by not allowing the reaction to proceed.
  • Mixed: Can bind to both E and ES; typically decreases Vmax, can affect Km depending on the affinity.
• Product Inhibition: Feedback mechanism where the product inhibits an earlier enzyme, maintaining homeostasis by regulating the flow through a pathway.

Metabolism Overview

• Two Main Types:
  • Catabolism: Breakdown of molecules to release energy, providing the necessary energy for cellular functions.
  • Anabolism: Uses energy to synthesize complex molecules, essential for growth, repair, and maintenance of cellular structures.
• Importance of ATP: Main energy currency in cells, produced primarily in the mitochondria via the Electron Transport Chain (ETC), underpinning many cellular processes.
• Energy Yield: Complete oxidation of glucose through glycolysis, the Krebs Cycle, and oxidative phosphorylation yields about 32 ATP, emphasizing the efficiency of cellular respiration.

Glycolysis and the Krebs Cycle

• Glycolysis Steps: 10 enzymatic steps occurring in the cytoplasm; breaks down glucose to pyruvate, yielding 2 ATP and 2 NADH, crucial for energy production in both aerobic and anaerobic conditions.
• Krebs Cycle (Citric Acid Cycle): Acetyl-CoA enters, yielding NADH, FADH₂, GTP (ATP), and CO₂; a key component of aerobic metabolism that provides electrons for the ETC.
• Electron Transport Chain (ETC): Functions by transferring electrons and driving ATP synthase for ATP production, with oxygen acting as the final electron acceptor, producing water as a byproduct.

Hormonal Regulation

• Insulin and Glucagon: Insulin promotes glucose uptake and storage, while glucagon triggers glycogen breakdown; together they help regulate blood sugar levels.
• Signaling Pathways: Use of second messengers like cAMP amplifies responses to hormonal signals, demonstrating the complexity of endocrine signaling in metabolic regulation.

Fasting & Metabolism Changes

• Metabolic Flexibility: The body shifts from glucose to fatty acids and ketone bodies for fuel during fasting, showcasing the adaptability of energy metabolism.
• Key Concepts in Starvation: The liver plays a crucial role in maintaining blood glucose via gluconeogenesis, synthesizing glucose from non-carbohydrate sources to support vital functions.

Conclusion

• Metabolism is a complex network: Enzyme activity regulation and hormonal control are critical to maintaining homeostasis and energy balance, illustrating the intricate interplay of biochemical processes in living organisms.