pp11 Metabolism Pathways

Metabolism Overview

• Final lecture in the metabolism segment of the course.

• Core metabolic network structure:

  • Comprises degradative pathways that convert organic compounds into intermediates.

  • Produces building blocks for macromolecules and secondary metabolites.

• Important concepts introduced include:

  • Autotrophs obtain carbon via CO₂ fixation.

  • Sources of nitrogen and energy through redox reactions or photosynthesis.

Definitions and Metabolic Pathways

• Metabolism: An entire system consisting of:

  • Catabolic pathways: Break down complex molecules into simpler ones.

  • Often involve oxidative reactions that generate:

    • NADH (reduced form of NAD⁺)

    • ATP (energy currency of the cell)

  • Results in materials for biosynthesis and energy for cell growth and reproduction.

  • Anabolic pathways: Involve biosynthesis and primarily utilize NADPH (produced by the conversion of NADH).

Learning Goals

• Usage of tools (Ecopsych or Metapsych) to explore details of metabolic pathways.

• Understanding importance of flux through metabolic pathways (speed of reactions).

• Analysis of how metabolic flux changes with environmental conditions.

Metabolic Network Visualization

• Complex diagram of metabolic pathways common across various organisms.

  • May initially be overwhelming but consists of recognizable patterns:

  • Cycles, pathways, and local networks.

TCA Cycle (Krebs Cycle)

• Central to carbon metabolism; connects carbohydrate metabolism to energy generation.

  • Acetyl CoA (from glycolysis or acetate) enters the cycle:

  • Produced CO₂ represents the loss of two carbons from acetyl group.

  • Generates:

    • Several molecules of NADH at three different points.

    • Flavin compound at a site generating fewer electrons.

    • One GTP (functionally analogous to ATP).

• Key intermediates serve as biosynthetic precursors:

  • Oxaloacetate → aspartate → methionine, lysine, threonine, isoleucine.

  • Alpha-ketoglutarate → glutamate → glutamine, arginine, proline, histidine.

  • Succinyl CoA is crucial for synthesizing lysine and methionine.

• Overall importance of TCA cycle: not only for energy but also for biosynthetic pathways.

Pathways Example: Pyridoxal 5'-Phosphate (PLP)

• Linear biosynthetic pathway studied in the lab.

• PLP is vitamin B6.

• Example shows both linear and branched pathways.

• Reference to EcoPsych database for details on E. Coli metabolic pathways.

Metabolic Quantification and Flux Analysis

• Metabolic reconstruction pioneered in 2007 allows understanding of metabolism as an entire system.

  • Estimated:

  • 1,200 metabolic reactions in the cytoplasm.

  • 192 reactions in the periplasm.

  • 8 extracellular reactions.

  • Transport reactions critical for substance movement across membranes:

  • 390 reactions transport compounds between cytoplasm and periplasm.

  • 298 from periplasm to extracellular space.

  • 2 from cytoplasm to outside cell.

  • 1,039 metabolites identified:

  • 951 in cytoplasm.

  • 418 in periplasm.

  • 299 secreted outside cell.

• Application of flux quantification has implications in drug targeting, understanding uncultivated microbes, and bioengineering for product synthesis.

Metabolite Concentrations in E. Coli

• 2009 study: first measurements of metabolite concentrations at exponential growth on glucose, showing significant variation (e.g., 96 mM glutamate vs. sub-micromolar adenosine).

• Snapshot view of concentrations does not reveal dynamic flux information.

Flux Omics

• Uses mass spectrometry to measure fluxes through metabolic pathways.

• Traces labeled carbon (e.g., using C-13) from a substrate fed to microbes.

  • Can determine where specific labels end up in metabolic products.

  • Predictive framework based on known pathways:

  • E.g., tracing C1 and C2 labeled glucose through glycolysis and pentose phosphate pathway.