- 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:
- 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 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
- Glycolysis
- ATP production
- Beta Oxidation
- Citric Acid Cycle
- Oxidative Phosphorylation
- Urea Cycle
- 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
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).