Metabolism Lecture Notes

INTRO TO METABOLISM

  • Essential Biochemistry is not just about breaking down molecules.
  • Sian Patterson, Ph.D., Associate Professor, Teaching Stream

Lecture Objectives

By the end of this lecture, students should be able to:

  • Compare the features of anabolic and catabolic pathways.
  • Review thermodynamics in the context of enzymes and pathways and predict favorable reactions.
  • Describe the importance of phosphoryl transfer potential for high energy molecules and reactions.
  • Describe the features of active sites that facilitate substrate binding, specificity, and enzyme catalysis.
  • Explain how enzymes can couple reactions to drive unfavorable reactions.
  • Explain the general organization and regulation of enzymes within a metabolic pathway.

Brain Break

  • What do you think of when you hear the term 'metabolism'?

Conflicts of Interest

  • The speaker has no conflict of interest to declare, nor is she a medical doctor (MD).
  • Viewers should make their own educated decisions about whether or not they need to take vitamins.

Catabolism and Anabolism are Interrelated

  • Catabolism:
    • Involves the breakdown of energy-yielding nutrients (carbohydrates, fats, proteins).
    • It is oxidative and exergonic, producing energy-poor end products (H₂O, CO₂, NH₃).
    • Generates chemical energy in the form of NADH, FADH₂, NADPH, and ATP.
  • Anabolism:
    • Involves the synthesis of cell macromolecules (proteins, polysaccharides, lipids, nucleic acids) from precursor molecules (amino acids, sugars, fatty acids, nitrogenous bases).
    • It is reductive and endergonic, requiring energy input (ATP, NADPH).

Metabolism

  • Metabolism is highly complex, involving numerous interconnected pathways.
  • These pathways include:
    • Glycosaminoglycan biosynthesis (e.g., chondroitin sulfate)
    • Nucleotide metabolism
    • Glycosphingolipid biosynthesis (lacto- and neolacto series, globoseries, ganglioseries)
    • Other glycan degradation
    • Proteoglycans
    • Isoflavonoid biosynthesis
    • Flavonoid biosynthesis (flavone and flavonol biosynthesis)
    • Linoleic acid metabolism
    • Anthocyanin biosynthesis
    • Monoterpenoid biosynthesis
    • Sesquiterpenoid and triterpenoid biosynthesis
    • Insect hormone biosynthesis
    • Diterpenoid biosynthesis
    • Phenylpropanoid biosynthesis
    • Carotenoid biosynthesis
    • Zeatin metabolism
    • Sphingolipid metabolism
    • Glycosylphosphatidylinositol (GPI)-anchor biosynthesis
    • beta-Alanine metabolism
    • Polyketide sugar metabolism
    • Pentose and glucuronate interconversions
    • Pyrimidine metabolism
    • Glycerophospholipid metabolism
    • Mannose metabolism
    • Carbohydrate metabolism
    • Lipid metabolism
    • Terpenoids and Polyketides metabolism
    • Photosynthesis
    • Amino Acid Metabolism
    • Energy Metabolism
    • Biosynthesis of type I polyketide products
    • Fatty acid metabolism and biosynthesis
    • Cutin, suberine and wax biosynthesis
    • Metabolism of other secondary metabolites

Metabolic Pathways

  • Glycolysis
  • ATP production
  • Beta Oxidation
  • Citric Acid Cycle
  • Oxidative Phosphorylation
  • Urea Cycle

Bioenergetics in Metabolism

  • Metabolism involves the catabolism of compounds, releasing free energy which can be ‘stored’ in ATP or other high energy intermediates.
  • Anabolic processes synthesize macromolecules using simple building blocks and energy.
  • The complex metabolic map can be broken down into linear, cyclic or even branched pathways.
  • Gibbs free energy (and enzymes) influence the metabolic flux or the conversion of metabolites through the pathways.

Chemical Reactions

  • Progress curve for a favorable reaction:
    • Includes the substrate, product, and transition state.
    • Shows the curve for when an enzyme is present.
    • Indicates where \Delta G is on the graph.

Gibbs Free Energy and Reactions

  • Gibbs Free Energy Change: \Delta G = \Delta Gº' + RT \ln \frac{[P]}{[S]}
    • \Delta G: Gibbs Free Energy Change
    • \Delta Gº': Standard Free Energy Change (1M, 298K, pH 7)
    • R: 8.314 J/mol*K
    • T: Temperature in Kelvin
    • [P]: Product concentration
    • [S]: Substrate concentration

Standard Free Energy Change

  • If K_{eq}' > 1, \Delta Gº' is negative, reaction is spontaneous.
  • If K_{eq}' < 1, \Delta Gº' is positive, reaction is non-spontaneous.
  • If K_{eq}' = 1, \Delta Gº' = 0, reaction is at equilibrium.
  • K_{eq}' = \frac{[P]}{[S]}
    • K_{eq}': Equilibrium constant

Actual Free Energy Change

  • Negative \Delta G: Spontaneous reaction
  • Positive \Delta G: Non-spontaneous reaction
  • Reactions can be reversible if \Delta G = 0.
  • The concentrations of substrates and products can influence directionality and overcome an unfavorable standard \Delta Gº'.
  • \Delta G = \Delta Gº' + RT \ln \frac{[P]}{[S]}

Thermodynamics and Biochemical Reactions

  • The Gibbs free energy under standard conditions (\Delta Gº' - 1M, 298K, pH 7.0) depends on the nature of the reactants and their concentrations at equilibrium (K'_{eq}).
  • The cellular Gibbs free energy (\Delta G) depends on the cellular concentrations of substrate and product and \Delta Gº'.
  • An enzyme can couple a favorable and unfavorable reaction to produce a new, overall favorable reaction.

Coupled Reactions (or Pathways)

  • Example:
    • A + B -> C ; \Delta Gº' = +14 \text{ kJ/mol}
    • C -> D ; \Delta Gº' = -31 \text{ kJ/mol}
    • Overall: A + B -> D ; \Delta Gº' = -17 \text{ kJ/mol}

ATP Phosphoryl Transfer Potential

  • ATP + H₂O -> ADP + Pi ; \Delta Gº' = -31 \text{ kJ/mol}
  • ADP + H₂O -> AMP + Pi ; \Delta Gº' = -34 \text{ kJ/mol}
  • AMP + H₂O -> Adenosine + Pi ; \Delta Gº' = -14 \text{ kJ/mol}
  • PPi + H₂O -> 2 Pi ; \Delta Gº' = -36 \text{ kJ/mol}
  • The hydrolysis of the different phosphoanhydride bonds have different free energies due to stabilization of the products.

ATP Structure

  • ATP consists of adenine, ribose, and three phosphate groups.
  • Hydrolysis of ATP to ADP releases energy (example: \Delta Gº' = -31 \text{ kJ/mol}).
  • Further hydrolysis of ADP to AMP releases additional energy (example: \Delta Gº' = -36 \text{ kJ/mol}).

Resonance Stabilization

  • Resonance structures contribute to the stability of phosphate compounds.

ATP Phosphoryl Transfer Potential

  • The change in free energy can be used to activate molecules for reactions that are highly unfavorable or found in anabolic pathways.
  • Major issue: how ATP and ADP are remade from ADP or AMP for future use.

Review - ATP

  • Nucleotide made of adenine, ribose, and three phosphates.
  • \alpha, \beta and \gamma phosphates: linked by phosphoanhydride bonds and an ester bond to the sugar.
  • Involved in both catabolic (breakdown) and anabolic (synthesis) pathways via phosphate group transfer reactions.
  • The change in free energy is dependent on the structures and differences in stability of the substrates vs. products.
  • Free energy changes are not fixed; the polar phosphates are ionizable and pH dependent, while metal ions (Mg2+) also help with stabilization.

Brain Break

  • What effect will the following have on the amount of free energy released?
    • Slight decrease in pH: decrease
    • Presence of Mg2+: increase

Compounds with Similar Phosphoryl Transfer Potentials

  • Example: Nucleoside diphosphokinase
    • GTP + ADP

Question

  • Rank the following in terms of importance of determining the flux through a metabolic pathway in a cell:
    • ATP availability
    • Changes in Gibbs free energy
    • The presence of enzymes
    • Metabolite concentration

Phosphoryl Transfer Potential

  • ATP’s function as a carrier of phosphoryl groups allows it to drive reactions that require an input of free energy.
  • The change in free energy \Delta Gº' arises due to the differences in free energies between the substrates and products.
  • Other molecules carry phosphates and have phosphoryl transfer potential or have bonds that release energy when broken.

Brain Break

  • Which ONE of the following molecules can be used to make ATP?
    • Phosphoenolpyruvate
    • Value is -62 kJ mol-1, which is the only one that is more negative than ATP to ADP + Pi (-32 kJ mol-1) as well as ATP to AMP + PPI (-45 kJ mol-1).

Coupled Reaction catalyzed by an enzyme

  • What is the coupled reaction catalyzed by the enzyme?
  • Phosphoenolpyruvate + H2O -> Pyruvate + Pi; \Delta Gº' = -62 \text{ kJ/mol}
  • ADP + Pi -> ATP + H2O; \Delta Gº' = +31 \text{ kJ/mol}
  • Net: ADP + Phosphoenolpyruvate -> ATP + Pyruvate; \Delta Gº' = -31 \text{ kJ/mol}

Example #2: Muscle Contraction

  • ATP + H2O -> ADP + Pi + H+ + energy
  • Catalyzed by ATPase

Phosphoryl Transfer Potential in Muscle

  • Phosphocreatine + ADP + H+
  • Catalyzed by Creatine kinase

Question

  • What is the standard free energy change for the phosphate transfer reaction catalyzed by creatine kinase, regenerating ATP?
    • A) - 43 kJ/mol
    • Stable, favourable, produces ATP

Creatine Kinase

  • Creatine Kinase Reversibility
  • ADP + Phosphocreatine
  • \Delta G_{overall} = \Delta Gº' + RT \ln \frac{[ATP][Creatine]}{[ADP][Phosphocreatine]}

Creatine Kinase

  • Phosphocreatine is a storage form of phosphates that can be used to make ATP in muscle.
  • Phosphocreatine has a larger, negative \Delta Gº' than ATP, which can be used to make ATP.
  • Creatine kinase catalyzes a coupled reaction and is controlled by Gibbs free energy changes.
  • Substrate and product concentrations can vary, driving the reaction in the reverse direction.

Regulation of a Pathway

  • A -> B -> C -> D -> E -> F
  • E1, E2, E3, E4, E5 are enzymes

Control of Flux

  • Metabolic flux is the conversion of metabolites through a pathway, under the assistance of enzymes.
  • An enzyme can speed up a reaction, but it is thermodynamics that dictates if a reaction will occur.
  • Large, negative free energy changes define the forward direction of a reaction or pathway.
  • Metabolite concentration and enzyme regulation can determine a pathway’s activity or directionality in a cell.

Lecture Summary

  • Metabolism consists of catabolic (energy producing) and anabolic (building) pathways.
  • Thermodynamics is the driving force behind the spontaneity of metabolic reactions.
  • The flux of the pathways can also be influenced by enzyme regulation, affecting how fast metabolites are made.
  • ATP and other high energy intermediates, as well as metabolite concentrations can drive unfavourable reactions in the forward direction, achieving the cell’s ultimate goal (catabolism vs. anabolism).