Enzymes and Metabolic Pathways

Enzymes

Introduction to Enzymes

• Enzymes are a diverse group of proteins that accelerate cellular reactions.

Chemical Reactions in Cells

• Chemical reactions occur when atoms possess sufficient energy to combine or alter bonding partners.
• Chemical bonds store potential energy; thus, chemical reactions involve energy changes.

Thermodynamics and Chemical Reactions

• The laws of thermodynamics govern all matter and energy transformations in the universe.
• First Law of Thermodynamics: Energy is neither created nor destroyed but can be transferred or transformed.
• Second Law of Thermodynamics: Disorder (entropy) tends to increase.
• Energy conversions result in some energy becoming unavailable for work.
• Energy transformations lead to increased disorder. Some energy is lost as random thermal motion (entropy).
• Life necessitates a constant energy input to maintain order.
• Reactions increasing entropy have more disordered products than reactants.
• Reducing disorder requires energy if there are fewer products than reactants.

Enthalpy vs. Entropy

• Enthalpy (H): Measures the total heat content in a thermodynamic system at constant pressure.
• Entropy (S): Measures the level of disorder in a thermodynamic system.

Relationship between Entropy, Enthalpy, and Gibbs Free Energy

• $ΔG = ΔH - TΔS$
  • Where:
    • $ΔG$ = Gibbs free energy
    • $ΔH$ = Enthalpy (in Joules or Kilojoules)
    • $T$ = Temperature (in Kelvin)
    • $ΔS$ = Entropy
• Gibbs Free Energy: The energy available to perform useful work.
• A reaction occurs spontaneously if ΔG < 0 (exergonic reaction).
• A reaction does not occur spontaneously if ΔG > 0 (endergonic reaction).

Free Energy

• Free Energy (G): The portion of a system’s energy available to do work under uniform temperature and pressure; combines enthalpy and entropy.
• Spontaneous processes decrease a system’s free energy, moving it to a more thermodynamically stable state.
• Decrease in free energy leads to an increase in disorder.

Energy Conversion in a Cell

• Example: Cellular respiration
  • Glucose + Oxygen → Carbon dioxide + Water + ATP
  • Fuel (Glucose) is converted into energy (ATP), producing waste products (Carbon dioxide and Water) and heat.

Change in Free Energy (ΔG)

• $ΔG = G<em>{\text{final state}} – G</em>{\text{initial state}}$
• -ΔG: Indicates the final product is at a lower energy state than the initial reactants.
  • Exergonic reaction
  • Spontaneous
  • Example: Cellular respiration, where $ΔG = -686 \text{ kcal/mol glucose}$
• +ΔG: Indicates the final product is at a higher energy state than the initial reactants.
  • Endergonic reaction
  • Non-spontaneous
  • Example: Photosynthesis, where $ΔG = 686 \text{ kcal/mol glucose}$

Exergonic vs. Endergonic Reactions

• $\Delta S$ = Change in entropy
• $\Delta H$ = Enthalpy change (heat evolved or absorbed at constant pressure)
• $\Delta G$ = Gibbs free energy change (measure of spontaneity)
• Spontaneity means a process occurs naturally without external work or energy.

Metabolism

• Metabolism: Sum of all chemical reactions in a biological system at a given time.
• Metabolism increases disorder (entropy).
• Example: Anabolic reactions to construct 1 kg of animal body require catabolism of about 10 kg of food.

Anabolic vs. Catabolic Reactions

• Anabolic Reactions: Link simple molecules to form complex ones.
  • Require energy input; energy is captured in chemical bonds.
  • Endergonic.
• Catabolic Reactions: Break down complex molecules into simpler ones.
  • Energy stored in chemical bonds is released.
  • Exergonic.

Catalysts and Enzymes

• Living systems depend on spontaneous reactions that occur slowly.
• Catalysts: Substances that speed up reactions without being permanently altered.
• Catalysts cannot make non-spontaneous reactions occur.
• Most biological catalysts are proteins called enzymes.
• Enzymes lower the activation energy, facilitating reactions.
• Example: Sucrose hydrolysis takes 15 days in solution; with sucrase, it takes 1 second.

Enzyme Specificity

• Enzymes are highly specific, catalyzing only one chemical reaction.
• Substrates: Reactants that bind to the enzyme's active site.
• Lock and Key Model: Enzyme specificity results from the precise 3-D shape and chemical properties of the active site.

Induced Fit Model

• Substrate binding induces a shape change in the enzyme for tighter binding.
• This shape change strains bonds in the substrate, leading to an unstable transition state.

Enzyme-Substrate Complex

• $E + S \rightarrow ES \rightarrow EP \rightarrow E + P$
• The enzyme-substrate complex (ES) is held together by hydrogen bonding, electrical attraction, or temporary covalent bonding.
• The enzyme remains unchanged after the reaction.

Mechanisms of Enzyme Catalysis

• Enzymes catalyze reactions through various mechanisms:
  • Increasing local substrate concentration.
  • Inducing strain on substrate bonds, creating an unstable transition state.
  • Orienting substrates for bond formation.
  • Adding chemical groups to the substrate.

Enzyme Partners

• Some enzymes require ions or other molecules (cofactors or coenzymes) to function.
  • Cofactors: Inorganic ions (e.g., Iron, Copper, Zinc).
  • Coenzymes: Organic molecules (e.g., Biotin, Coenzyme A, NAD, FAD, ATP).
  • Prosthetic Groups: Heme, Flavin, Retinal

Rate of Reaction

• Rate of reaction = amount of product produced in a given period of time

• Uncatalyzed Reaction: Rate is directly proportional to substrate concentration.

• Catalyzed Reaction: Rate levels off when the enzyme is saturated due to less enzyme than substrate.

• Saturated means all enzymes are working at their maximum rate.

• Maximum reaction rate varies from 1 to 40 million molecules per second.

  calculating rate of reaction.

• Rate = $\frac{dY}{dt}$

  • Where $\frac{dY}{dt}$ = $\frac{amount \, of \,change}{change \,in \,time}$

• Example: Catalase breaking down hydrogen peroxide:

  • From 2 to 4 minutes, if oxygen production changes from 3.6 mL to 5.5 mL:
    • Rate = $\frac{5.5 - 3.6}{4 - 2} = 0.95 \text{ mL/min}$

Factors Affecting Enzyme Reaction Rate

• Changing Substrate Concentration
• Changing Enzyme Concentration
• Changing Temperature
• Changing pH

Temperature

• Warming increases reaction rates up to a point.
• High temperatures can break non-covalent bonds and inactivate enzymes (denaturation).
• Enzymes have optimal temperatures for activity.

pH

• Protein tertiary structure (and function) is sensitive to pH.
• Enzymes have optimal pH levels for activity.

Isozymes

• Isozymes catalyze the same reaction but have different compositions and physical properties.
• They may have different optimal temperatures or pH levels, allowing adaptation to environmental changes.

Metabolic Pathways

• Enzyme-catalyzed reactions occur in metabolic pathways where the product of one reaction is the substrate for the next.
• Homeostasis depends on the activity of these enzymes.
• Cells regulate metabolic pathways by:
  • Regulating the amount of enzyme.
  • Regulating enzyme activation.

Regulation of Metabolic Pathways

• Chemicals either activate or inhibit enzymes.
• Enzymes are controlled by:
  • Allosteric regulation (activation or inhibition/noncompetitive inhibition).
  • Competitive inhibition.

Allosteric Regulation

• A non-substrate molecule binds to a site other than the active site.
  • Binding changes the active site shape, either opening or closing it to the substrate.
• Allosteric Inhibition: Binding closes the active site; the substrate cannot bind, temporarily turning off the enzyme.
• Allosteric Activation: Binding opens the active site, allowing the substrate to bind, temporarily turning on the enzyme.

Enzyme Modification

• Allosteric regulation can be achieved by adding or removing functional groups.
• Kinases: Enzymes that add phosphate groups to activate or inhibit other enzymes.
• Phosphatases: Enzymes that remove phosphate groups, reversing kinase activity.

End-Product Inhibition (Negative Feedback)

• Plays an important role in controlling metabolic pathways.
• When a pathway produces a specific product, the product inhibits the pathway.
• Excess end products interact with enzymes early in the pathway, decreasing its activity.
• When end products are used up, inhibition is released, allowing the pathway to function again.
  This process results in production of more end products.

Enzyme Inhibition

• Enzyme inhibition is caused by chemicals interfering with enzyme function

Metabolic Regulation

• Cells use natural inhibitors or activators to regulate metabolic pathways.
• Activator or inhibitor binding is reversible, held together by weak attractions.
• The cell can remove the activator or inhibitor to adjust pathway speed.
• Example: Threonine deaminase converts threonine to isoleucine.
  • Excess isoleucine binds to an allosteric site on threonine deaminase, decreasing its activity and inhibiting isoleucine production.
  • When isoleucine is scarce, it is released from the allosteric site, and threonine deaminase resumes activity.

Competitive Inhibition

• A molecule other than the substrate binds to the active site.
  • It competes with the natural substrate and blocks it from binding.
• There is no competitive activation.
• Can be overcome by increasing the substrate concentration.

Irreversible Inhibition

• Inhibitor covalently binds to a side chain in the active site.
• The enzyme is permanently inactivated.
• Example: Poison or toxin DIPF (Diisopropyl fluorophosphate).
• DIPF forms a stable covalent bond with serine at the active site of acetylcholinesterase, disabling the enzyme.

Noncompetitive Inhibition

• Cannot be overcome by increasing substrate concentration.

Feedback Inhibition

• The final product acts as a noncompetitive inhibitor of the first unique enzyme, shutting down the pathway.

Feedback Inhibition Example: Isoleucine Synthesis

Step by Step Explanation of Feedback Inhibition:

• The initial substrate (threonine) needs to convert to end product (isoleucine) through series of steps.
• Threonine, in active site of enzyme 1(threonine deaminase will lead to intermediate A.
• When isoleucine is used up by cell it freely binds to allosteric site.
• Active site will no longer bind, pathway is switched off.
• Process repeats until isoleucine builds up enough to repeat process.

Feedback Inhibition Example: Glycolysis

• The presence of ATP at the end of the pathway inhibits the phosphofructokinase enzyme, lowering
  the affinity for fructose-6-phosphate and slowing
  down the glycolysis pathway. Phosphofructokinase
  aids the addition of a phosphate group, changing
  fructose-6-phsophate to fructose-1,6-bisphosphate.
• This is an example of feedback inhibition because a
  product at the end of the metabolic pathway inhibits
  an enzyme in the beginning of the pathway.

Aspirin as an Enzyme Inhibitor

• Aspirin binds to and inhibits the enzyme cyclooxygenase (COX).
• Cyclooxygenase catalyzes the commitment step for metabolic pathways that produce:
  • Prostaglandins—involved in inflammation and pain
  • Thromboxanes—stimulate blood clotting and constriction of blood vessels
• Aspirin binds at the active site of cyclooxygenase and transfers an acetyl group to a serine residue.
• Serine becomes more hydrophobic, which changes the shape of the active site and makes it inaccessible to the substrate.

Protein Denaturation

• Protein structure depends on physical and chemical conditions
• Denaturation – When the chemical bonds and interactions of a protein are destroyed, causing the protein to unravel and lose its shape
• Caused by: pH, salt concentration, and temperature

Origin of Life Experiment

• One way to investigate the possibility of life on other planets is to study how life may have originated on Earth.
• An experiment in the 1950s combined gases thought to be present in Earth’s early atmosphere, including water vapor. An electric spark provided energy.
• Complex molecules were formed, such as amino acids. Water was essential in this experiment.