Metabolism and its control W10

Regulation of Metabolism

  • Introduction by Dr. C Montiel-Duarte.

Learning Outcomes

  • K2: Discuss the roles of enzymes, substrate levels, energy change, and compartmentalisation in regulating flux through metabolic pathways.

  • K3: Explain the roles of hormones in regulating and integrating metabolism in humans.

    • References:

    • Chapter 3, Biochemistry, 6th Ed.

    • Chapter 15, Principles of Biochemistry, 5th Ed.

Studying Metabolism

  • Purification of Proteins:

    • Protein must be released from the cell.

    • Purification assays are required to identify the protein:

    • Enzyme Activity: Evaluates the impact of the enzyme.

    • Specific Activity: Measure of purity.

Methods to Study Metabolism

  1. Subcellular Fractionation:

  2. In Vivo Studies Using Radioisotopes

  3. Case Study:

    • Comparing brain metabolism in a normal subject to a cocaine addict using 18FDG/PET imaging.

    • Observation: Cocaine addicts show depressed metabolism in frontal brain regions, potentially linking to loss of control.

  4. Protein Purification Techniques:

    • Techniques include solubility, charge, size, and binding abilities.

    • Amino Acid Sequencing using Edman degradation.

    • Determining 3D structure through X-ray crystallography identifies active sites.

    • Location-validation performed using antibodies.

Homeostasis

  • Definition: Maintenance of internal stability.

  • Deregulation Consequences: Can lead to disease or death.

  • Enzyme Activity Regulation: Essential for metabolic pathways; must be modulated to avoid energy waste.

Regulatory Strategies

  • General Methods: Control the number of enzymes and their activities.

  • Key Steps in Regulation:

    1. Extracellular signal activates the receptor.

    2. Regulatory protein interacts with the enzyme, leading to:

    • Phosphorylation or dephosphorylation.

    • Transcription of specific genes.

    • mRNA degradation and translation on ribosomes.

    • Protein degradation via the ubiquitin-proteasome system or lysosomal autophagy.

Control Over Enzyme Amounts

  • Techniques and Methods:

    • Utilize techniques like microarray and RNA-Seq for gene expression analysis comparing samples.

    • Controls include:

    • mRNA degradation.

    • Proteasome system.

    • RNA isolation and RT-qPCR analysis.

Control of Enzyme Activity

  • Key Techniques:

    1. Allosteric Control:

    • Enzymes have distinct regulatory and functional sites. Shared activity can be affected by cooperativity.

    1. Multiple Forms of Enzymes (Isoenzymes):

    • Variants with slight differences lead to different kinetic properties (e.g., different km and Vmax).

    1. Covalent Modifications:

    • Often reversible modifications like phosphorylation affect enzyme localization and activity.

    1. Proteolytic Activation:

    • Activation processes such as converting zymogens to active enzymes (e.g., blood clotting factors, digestive enzymes).

Reflection on Purification Techniques

  • Researchers sometimes find increased total activity during purification, suggesting mechanisms of enzyme regulation may enhance functionality of concentrated enzymes.

Summary

  • Protein purification is crucial for studying enzymatic functions.

  • Multiple regulatory strategies exist to control both the quantity and activity of enzymes, ensuring metabolism is adequately controlled.