Regulation of metabolism

• Protein Release


  Cell lysis (mechanical, chemical, enzymatic)
• Protein Purification Steps


  1. Centrifugation

  2. Chromatography (ion exchange, affinity, size exclusion)

  3. Electrophoresis
• Assays


  Enzyme assays to measure catalytic activity, specific assays for target protein identification
• Specific Activity


  Ratio of enzyme activity to the amount of protein, indicating enzyme purity
• Subcellular Fractionation


  Differential centrifugation to separate cellular components based on size and density
• In Vivo Studies


  Use of radioisotopes (e.g., 14C, 32P) to trace metabolic pathways
• Protein Characteristics


  • Solubility
  • Charge
  • Size
  • Binding Abilities
• Amino Acid Sequencing


  Edman Degradation: Sequentially removes and identifies N-terminal amino acids of a protein
• Protein Localization


  Use of Antibodies: Immunocytochemistry to visualize protein distribution in cells
• 3D Structure Analysis


  1. ray Crystallography: Provides detailed structure of protein's active sites and interactions
• Homeostasis


  The ability of an organism to maintain a stable internal environment despite external changes
• Importance of Homeostasis


  • Essential for survival, as enzymes and metabolic pathways need precise conditions (pH, temperature, ion concentrations)
• Deregulation of Homeostasis


  Results in metabolic disorders (e.g., diabetes, metabolic syndrome), leading to disease or death
• Regulatory Strategies


  • Control of Enzyme Amount
  • Control of Enzyme Activity
• Control of Enzyme Amount

  1. Gene Expression: Regulation at transcriptional (gene transcription) and post-transcriptional (mRNA stability, translation) levels

  2. Protein Degradation: Ubiquitin-proteasome system targets proteins for degradation
• Control of Enzyme Activity

  • Allosteric Control
  • Feedback Inhibition
  • Multiple Forms of Enzymes (Isoenzymes)
  • Covalent Modifications
  • Localization and Degradation
  • Proteolytic Activation
• Allosteric Control


  • Regulatory Sites: Binding sites separate from the active site where molecules can increase or decrease enzyme activity
  • Cooperativity: Binding of a substrate to one active site affects activity at other sites (e.g., hemoglobin)
• Feedback Inhibition


  End product of a pathway inhibits an enzyme involved early in the pathway
• Multiple Forms of Enzymes (Isoenzymes)

  • Structural Differences: Small variations in amino acid sequences
  • Kinetic Properties: Differences in KM (affinity for substrate) and Vmax (maximum rate)
  • Tissue-Specific Expression: Different tissues may express different isoenzymes for the same reaction
• Covalent Modifications


  • Reversible Modifications: Phosphorylation, acetylation, methylation, ubiquitination
  • Activation/Inhibition: Modifications can either activate or inhibit enzyme function
  • Localization: Enzymes may be directed to specific cellular compartments
  • Degradation: Targeted degradation of enzymes via lysosomes or proteasomes
• Proteolytic Activation


  Zymogens/Proenzymes: Inactive precursors that require proteolytic cleavage to become active
• Proteolytic Activation Examples


  • Blood Clotting: Activation of clotting factors
  • Digestive Enzymes: Activation of pepsinogen to pepsin
  • Programmed Cell Death (Apoptosis): Activation of caspases
• Allosteric Control Mechanism


  Non-substrate molecules bind to allosteric sites, inducing conformational changes affecting enzyme activity
• Allosteric Control Example


  • Aspartate transcarbamoylase (ATCase) is regulated by CTP (inhibitor) and ATP (activator)
• Multiple Forms of Enzymes (Isoenzymes)

  Fine-Tuning Metabolism: Allows cells to adapt to different metabolic needs
• Isoenzyme Example


  • Hexokinase and glucokinase are isoenzymes that phosphorylate glucose but have different kinetic properties and tissue distributions
• Covalent Modifications: Phosphorylation


  Catalyzed by kinases and reversed by phosphatases
• Phosphorylation Example


  • Glycogen phosphorylase is activated by phosphorylation during glycogen breakdown
• Proteolytic Activation


  One-Time Activation: Irreversible activation mechanism
• Proteolytic Activation Example


  • Trypsinogen is activated to trypsin in the digestive tract, which then activates other digestive enzymes
• Protein Purification is necessary for functional and structural studies
• Enzyme Regulation is essential for efficient and controlled metabolic processes
• Regulatory Strategies


  • Controlling enzyme amounts
  • Controlling enzyme activity through allosteric regulation
  • Isoenzymes
  • Covalent modifications
  • Proteolytic activation