Chapter 6: Energy, Enzymes, and Metabolism

This chapter provides a foundational understanding of energy transformations, the role of enzymes as biological catalysts, and the intricate network of metabolic reactions that sustain life. It begins with a practical example of how common medications like aspirin and ibuprofen function by inhibiting specific enzymes, then delves into the fundamental principles governing chemical reactions, the nature of energy, and the complex regulation of cellular metabolism.

Introduction to Energy, Enzymes, and Metabolism

Aspirin and Ibuprofen as Enzyme Inhibitors
- Historically, pain relief came from willow tree bark (salicylic acid).
- Modern aspirin uses acetylsalicylic acid, a gentler derivative.
- These drugs work by inhibiting the enzyme cyclooxygenase.
- Cyclooxygenase is essential for synthesizing prostaglandins, molecules involved in inflammation, pain, and fever.
- By inhibiting cyclooxygenase, aspirin and ibuprofen block prostaglandin production, thereby relieving pain and fever.
Enzymes as Biological Catalysts
- Enzymes are proteins that significantly speed up thousands of different chemical reactions within cells.
- A chemical reaction involves changing one or more substances into others, which can include:
  - Molecules combining to form larger molecules.
  - Molecules breaking apart into smaller ones.
  - Rearrangements of atoms within molecules.
  - Transfer of electrons between atoms.
- Living cells continuously perform thousands of these reactions to maintain life.
Metabolism Defined
- Metabolism is the sum total of all chemical reactions occurring within an organism.
- It also refers to specific sets of cellular chemical reactions (e.g., sugar metabolism, fat metabolism).
- Most metabolic processes involve either the breakdown or synthesis of organic molecules.
- Organic molecules are crucial for maintaining cell structure, providing building blocks, and storing chemical energy to power cellular processes.
Chapter Overview
- General discussion of energy and chemical reactions.
- Factors controlling reaction direction and rate, with a focus on enzymes.
- Cellular metabolism, including coordinated metabolic pathways.
- Regulation of metabolic pathways and recycling of organic molecules.

6.1 Energy and Chemical Reactions

Two primary factors dictate the fate of a chemical reaction in a living cell: its direction and its rate.

Generalized Chemical Reaction
- Represented as: aA + bB \rightleftharpoons cC + dD
- A and B are reactants, C and D are products.
- a, b, c, d are the stoichiometric coefficients (moles).
- This reaction is reversible, meaning it can proceed in either the forward (A+B to C+D) or reverse (C+D to A+B) direction.
- The direction depends on energy considerations and the concentrations of reactants and products.
- Cells utilize energy intermediate molecules, such as ATP, to drive reactions in desired directions.
Energy Exists in Different Forms
- Energy is defined as the ability to cause change or do work.
- Kinetic energy: Energy associated with movement (e.g., a baseball bat in motion).
- Potential energy: Energy a substance or object possesses due to its structure or location.
  - Example: An electron in an outer shell has higher potential energy than one in an inner shell. When it drops to a lower shell, potential energy converts to kinetic energy.
- Chemical potential energy (chemical energy): Energy stored in atoms and the bonds between atoms, released during chemical reactions.
  - Organic molecules like glucose store significant chemical energy.
  - Breakdown of glucose releases energy to synthesize energy intermediates like ATP.
- Thermodynamics: The study of energy interconversions.
- Laws of Thermodynamics:
  - First Law of Thermodynamics (Law of Conservation of Energy):
    - Energy cannot be created or destroyed.
    - Energy can be transferred from one place to another and transformed from one type to another (e.g., chemical energy to heat).
  - Second Law of Thermodynamics:
    - Any energy transfer or transformation increases the degree of disorder of a system, known as entropy (S).
    - Entropy is a measure of molecular randomness. Increased disorder means increased entropy.
    - As energy becomes more evenly distributed, it becomes less able to promote change or do work.
    - Some energy may become unusable by living organisms during conversion (e.g., unusable heat released by a chemical reaction).
The Change in Free Energy Determines the Direction of a Chemical Reaction
- Cells require energy for chemical reactions, cellular movements (e.g., muscle contraction), and maintaining organization.
- Distinction between usable and unusable energy:
  - Total energy = Usable energy + Unusable energy.
  - Unusable energy is primarily due to entropy, which cannot be harnessed.
- Enthalpy (H): The total energy of a system.
- Free energy (G): The usable energy, available to promote change or do work (named after J. Willard Gibbs).
- Entropy (S): The amount of unusable energy in a system.
- Relationship: H = G + TS (where T is absolute temperature in kelvins).
- Rearranged for free energy: G = H – TS.
- Spontaneity of Reactions:
  - A critical question is whether a process occurs spontaneously (e.g., glucose breakdown).
  - A spontaneous reaction does not necessarily mean it is fast; its rate can be very slow.
- Free-Energy Change (ΔG):
  - The key to evaluating spontaneity is the free-energy change: \Delta G = \Delta H – T\Delta S.
  - Δ indicates a change before and after a reaction.
  - Exergonic reactions:
    - Have a negative free-energy change (ΔG < 0).
    - Products have less free energy than reactants.
    - Free energy is released during product formation.
    - These reactions are spontaneous.
  - Endergonic reactions:
    - Have a positive free-energy change (ΔG > 0).
    - Require the addition of free energy.
    - These reactions are not spontaneous.
  - A negative ΔG favors product formation; a positive ΔG favors reactant formation.
  - ATP Hydrolysis Example:
    - Adenosine triphosphate (ATP) is a common energy source.
    - Hydrolysis of ATP: ATP is broken down into adenosine diphosphate (ADP) and inorganic phosphate (Pi) using water.
    - \Delta G for ATP hydrolysis is -7.3 kcal/mol, indicating a strongly favored, spontaneous reaction that releases energy.
Cells Use ATP to Drive Endergonic Reactions
- Many biological processes are endergonic and non-spontaneous.
- Cells overcome this by coupling exergonic reactions with endergonic reactions.
- An endergonic reaction will proceed spontaneously if the combined net free-energy change (ΔG) for both coupled processes is negative.
- Example: Glucose Phosphorylation
  - Endergonic reaction: Glucose + Phosphate2− → Glucose-6-phosphate2− + H2O (ΔG = +3.3 kcal/mol)
  - Exergonic reaction: ATP4− + H2O → ADP2− + Pi2− (ΔG = -7.3 kcal/mol)
  - Coupled reaction: Glucose + ATP4− → Glucose-6-phosphate2− + ADP2− (Net ΔG = -4.0 kcal/mol)
  - In this coupled reaction, a phosphate is directly transferred from ATP to glucose, a process called phosphorylation.
  - The negative net ΔG makes the coupled reaction spontaneous.
- ATP Recycling:
  - Human cells use millions of ATP molecules per second.
  - The breakdown of food molecules (exergonic) releases energy to synthesize ATP from ADP and phosphate (endergonic).
  - This recycling is rapid: an average person hydrolyzes ~100 pounds of ATP daily, with each ATP molecule undergoing ~10,000 cycles of hydrolysis and regeneration.
Genomes Encode Many Proteins That Use ATP as a Source of Energy
- Many proteins utilize ATP hydrolysis to drive chemical reactions or cellular processes.
- Biochemists have identified specific amino acid sequences within proteins that function as ATP-binding sites.
- This allows researchers to predict if a newly discovered protein uses ATP.
- Genomic analysis (proteomes) reveals that, on average, over 20% of all proteins bind ATP across bacteria, archaea, and eukaryotes.
- In humans, with ~22,000 protein-coding genes, at least 4,400 genes encode ATP-utilizing proteins, highlighting ATP's immense importance.

6.2 Enzymes and Ribozymes

Most cellular chemical reactions require a catalyst to proceed at a rapid pace.

Catalysts and Enzymes
- A catalyst is an agent that speeds up a chemical reaction without being permanently changed or consumed.
- In living cells, the most common catalysts are enzymes, which are proteins.
- The term "enzyme" was coined by Wilhelm Kuhne in 1876, following his discovery of trypsin.
Enzymes Increase the Rates of Chemical Reactions
- Thermodynamics determines reaction direction (spontaneity), but not its rate.
- Enzyme-catalyzed reactions are typically millions of times faster than uncatalyzed reactions.
  - Example: Catalase speeds up H2O2 breakdown 1015-fold.
- Chemical reactions involve bond breaking and forming, which requires straining/stretching bonds or proper molecular positioning.
- Activation Energy (EA):
  - An initial input of energy required for molecules to collide in the correct orientation and possess enough energy to overcome electron repulsion and rearrange bonds.
  - Allows molecules to reach a transition state, where original bonds are stretched to their limit, enabling product formation.
  - The activation energy barrier is why many reactions are slow.
  - Enzymes lower this activation energy, allowing reactions to proceed rapidly with available heat.
- How Enzymes Lower Activation Energy:
  - Straining reactants: Enzymes bind reactants, straining their bonds and making it easier to reach the transition state.
  - Positioning reactants close together: For reactions with multiple reactants, enzymes provide a site where reactants are brought into close proximity and optimal orientation, facilitating new covalent bond formation.
Enzymes Recognize Their Substrates with High Specificity and Undergo Conformational Changes
- Active site: The specific location on an enzyme where the chemical reaction occurs.
- Substrates: The reactant molecules that bind to the enzyme's active site and participate in the reaction.
  - Example: For hexokinase, glucose and ATP are substrates.
- Enzyme-substrate complex: Formed when the enzyme binds to its substrate(s).
- High Specificity: Enzymes typically bind their substrates with high specificity (e.g., hexokinase recognizes glucose but not fructose or galactose well).
  - Lock-and-key model (Emil Fischer, 1894): Proposed that enzyme-substrate recognition is like a specific key (substrate) fitting into a specific lock (active site).
- Induced Fit (Daniel Koshland, 1958):
  - Further research showed that enzyme-substrate interaction involves conformational changes (movements) in the enzyme itself.
  - These changes cause substrates to bind more tightly to the enzyme.
  - Induced fit is a key phenomenon that further lowers the activation energy.
  - The enzyme catalyzes the reaction only after induced fit has occurred.
Enzyme Function Is Influenced by the Substrate Concentration and by Inhibitors
- Affinity: The degree of attraction between an enzyme and its substrate(s).
  - High affinity: Enzyme binds substrates even at low concentrations.
  - Low affinity: Enzyme-substrate complex forms only at higher substrate concentrations.
- Substrate Concentration and Reaction Velocity:
  - As substrate concentration increases, reaction velocity (rate of product production) increases.
  - Eventually, the velocity reaches a plateau because the enzyme becomes saturated with substrate (nearly all active sites are occupied).
  - Vmax: The maximal rate of the chemical reaction when the enzyme is saturated.
  - KM (Michaelis constant): The substrate concentration at which the reaction velocity is half its maximal value (Vmax/2).
    - A measure of the substrate concentration required for a reaction to occur.
    - High KM means higher substrate concentration is needed for a given velocity.
  - Enzyme-catalyzed reaction steps: E + S ⇌ ES → E + P (E=enzyme, S=substrate, ES=enzyme-substrate complex, P=product).
  - Relationship between KM and Affinity: If product formation is much slower than substrate release, KM is inversely related to affinity.
    - High KM = low affinity (weak binding).
    - Low KM = high affinity (strong binding).
- Enzyme Inhibitors: Molecules that decrease enzyme function.
  - Reversible Inhibitors: Bind noncovalently and can dissociate. Cells commonly use these to modulate enzyme function.
    - Competitive Inhibitors:
      - Bind noncovalently to the active site, competing with the substrate.
      - Often mimic the substrate's structure.
      - Increase the KM for the substrate (more substrate needed to reach Vmax/2).
      - Their effects can be overcome by increasing substrate concentration.
    - Noncompetitive Inhibitors:
      - Bind noncovalently to an allosteric site (a location outside the active site).
      - Lower the Vmax of the reaction.
      - Do not affect the KM.
  - Irreversible Inhibitors:
    - Usually bind covalently to an enzyme, permanently inactivating its function.
    - Example: Diisopropyl phosphorofluoridate (DIFP), a nerve gas, covalently reacts with acetylcholinesterase, crucial for neuron function.
    - Not a common cellular control mechanism because permanent inactivation is energy-inefficient (enzyme cannot be reused).
Additional Factors Influence Enzyme Function
- Nonprotein Components: Enzymes sometimes require nonprotein molecules or ions.
  - Prosthetic groups: Small molecules permanently attached to the enzyme surface, aiding function.
  - Cofactors: Inorganic ions (e.g., Fe3+, Zn2+) that temporarily bind and promote a reaction.
  - Coenzymes: Organic molecules that temporarily bind, participate in the reaction, but are unchanged at completion.
- Environmental Factors:
  - Temperature: Most enzymes have an optimal temperature range.
    - Human enzymes often function best at 37°C.
    - Temperatures significantly above or below optimal inhibit function.
    - Very high temperatures can denature proteins, causing them to unfold and lose their 3D shape and function.
  - pH: Enzyme function is sensitive to pH.
    - Pepsin (stomach protease) functions optimally at pH ~2.0 (highly acidic).
    - Many cytosolic enzymes function optimally at a more neutral pH, such as pH 7.2.
    - Significant deviations from optimal pH decrease enzyme function.
  - Ionic conditions also play a role.
Feature Investigation: The Discovery of Ribozymes by Sidney Altman Revealed That RNA Molecules May Also Function as Catalysts
- Historical Context: Until the 1980s, all biological catalysts were thought to be proteins.
- RNase P Study:
  - RNase P is a catalyst involved in processing tRNA molecules (precursor tRNA, ptRNA, into mature tRNA).
  - It cleaves a covalent bond at a specific site in ptRNA, releasing a 5' fragment.
- Altman's Discovery:
  - Sidney Altman and colleagues purified RNase P from E. coli.
  - They found it had two subunits: an RNA molecule (377 nucleotides) and a small protein (14 kDa). This was unexpected for a catalyst.
- Cecilia Guerrier-Takada's Experiment (Altman's lab):
  - Investigated the effects of magnesium ion (Mg2+) on RNase P function in vitro.
  - Compared ptRNA processing at low (10 mM MgCl2) and high (100 mM MgCl2) magnesium concentrations.
  - Samples incubated with: no RNase P (control), RNA subunit alone, or intact RNase P (RNA + protein subunits).
  - Gel electrophoresis was used to detect cleaved tRNA.
  - Results:
    - Controls (no RNase P) showed no processing.
    - At low MgCl2, RNA subunit alone showed no processing; intact RNase P did.
    - Crucial finding: At high MgCl2, the RNA subunit alone was sufficient to cleave ptRNA into tRNA and a 5' fragment.
  - Interpretation: High MgCl2 likely helps the RNA subunit maintain a catalytically active conformation, similar to the role of the protein subunit in a living cell.
- Conclusion: Ribozymes:
  - Subsequent work confirmed the RNA subunit of RNase P is a true catalyst.
  - Around the same time, Thomas Cech and colleagues independently discovered catalytic activity in an RNA molecule from Tetrahymena thermophila.
  - The term ribozyme was coined for RNA molecules that catalyze chemical reactions.
  - Altman and Cech received the Nobel Prize in Chemistry in 1989 for this discovery.
  - Ribozymes are now known to play key catalytic roles in cells.

6.3 Overview of Metabolism

In living cells, individual chemical reactions are coordinated and often occur in a series of steps called a metabolic pathway, with each step catalyzed by a specific enzyme.

Categories of Metabolic Pathways:
- Catabolic reactions:
  - Break down larger molecules into smaller ones.
  - Often exergonic (release energy).
- Anabolic reactions:
  - Synthesize larger molecules from smaller precursor molecules.
  - Usually endergonic (require energy input) and must be coupled to exergonic reactions in cells.
Catabolic Reactions Recycle Organic Building Blocks and Produce Energy Intermediates Such as ATP
- Two main uses of catabolic reactions:
  1. Recycling of Organic Building Blocks:
    - Break down macromolecules into their constituent organic molecules (monomers).
    - These monomers are then reused as building blocks for new molecules and macromolecules.
    - Example: Proteases break peptide bonds in improperly folded or unneeded proteins, generating amino acids for new protein synthesis.
  2. Breakdown of Organic Molecules to Obtain Energy:
    - Covalent bonds in organic molecules (e.g., glucose) store significant energy.
    - Cells do not directly use this released energy; instead, it's stored in energy intermediates like ATP.
    - ATP is then directly used to drive endergonic reactions.
    - Example: Glycolysis (breakdown of glucose to pyruvate):
      - A catabolic pathway where energy released from bond breakage in glucose is harnessed to synthesize ATP.
      - Involves a series of steps with bond rearrangements.
      - Produces molecules that readily donate a phosphate group to ADP.
      - Example: Phosphoenolpyruvate has an unstable phosphate bond, which can be transferred to ADP to form ATP and pyruvate (ΔG = -7.5 kcal/mol, exergonic).
    - Substrate-level phosphorylation: A way of synthesizing ATP where an enzyme directly transfers a phosphate from an organic molecule to ADP.
    - Chemiosmosis: Another mechanism for ATP synthesis, using energy stored in an ion electrochemical gradient (discussed in Chapter 7).
Redox Reactions Involve the Transfer of Electrons
- Oxidation: The removal of one or more electrons from an atom or molecule.
  - Often involves oxygen.
  - A substance that has been oxidized generally has less energy.
- Reduction: The addition of one or more electrons to an atom or molecule.
  - Named because adding a negatively charged electron reduces the net charge.
  - A substance that has been reduced generally has more energy.
- Redox reaction (reduction-oxidation reaction):
  - Electrons are not free in solution; when one molecule is oxidized, another must be reduced.
  - Generalized equation: Ae^{-} + B \rightarrow A + Be^{-} (A is oxidized, B is reduced).
- NADH Formation:
  - During the oxidation of organic molecules (e.g., glucose), removed electrons can be used to produce energy intermediates like NADH.
  - In this process, an organic molecule is oxidized, and nicotinamide adenine dinucleotide (NAD+) is reduced to NADH.
  - Uses of NADH:
    - Oxidation of NADH is highly exergonic and can be used to make ATP (Chapter 7).
    - NADH can donate electrons to other organic molecules, energizing them to more readily form covalent bonds, crucial for anabolic reactions.
Anabolic Reactions Require an Input of Energy to Make Larger Molecules
- Also called biosynthetic reactions.
- Essential for synthesizing larger molecules and macromolecules (e.g., RNA, proteins, amino acids, fats).
- Involve forming covalent linkages between precursor molecules.
- Require energy input, often supplied by energy intermediates like NADH.
  - Example: Glutamate (amino acid) synthesis from α-ketoglutarate and ammonium (NH4+) requires NADH.
Metabolic Pathways Are Regulated in Three General Ways
- Regulation is crucial to ensure organic molecules are broken down only when needed for energy or recycling, and synthesized only when required.
- Levels of Regulation:
  1. Gene Regulation:
    - Enzymes are proteins encoded by genes.
    - Cells control metabolic pathways by turning genes encoding specific enzymes on or off.
    - Example: Bacteria turn off genes for sugar-breaking enzymes if that sugar is absent, and turn them on when it becomes available.
  2. Regulation via Cell-Signaling Pathways:
    - Cells integrate environmental signals to adjust metabolism.
    - Cell-signaling pathways often activate protein kinases (enzymes that phosphorylate target proteins).
    - Example: Epinephrine (fight-or-flight hormone) binds to muscle cells, stimulating an intracellular pathway that phosphorylates enzymes involved in carbohydrate metabolism.
    - This promotes carbohydrate breakdown, providing energy for fight-or-flight.
    - When fright subsides, phosphatases remove phosphate groups, restoring normal metabolism.
  3. Biochemical Regulation:
    - Involves the noncovalent binding of a molecule directly to an enzyme, regulating its function.
    - Includes competitive and noncompetitive inhibition (as discussed earlier).
    - Feedback Inhibition:
      - A common type of noncompetitive inhibition.
      - The product of a metabolic pathway inhibits an enzyme acting early in the pathway.
      - Prevents overaccumulation of the product.
      - This regulation is reversible; inhibition occurs only when product concentration is high.
      - Enzymes regulated by feedback inhibition often have two binding sites: an active site and an allosteric site.
      - Binding of the product to the allosteric site causes a conformational change in the enzyme, inhibiting its catalytic function (e.g., altering the active site).
      - Allosteric sites are typically found in enzymes catalyzing early, rate-limiting steps.
- Regulation of the Rate-Limiting Step:
  - In a metabolic pathway with multiple enzyme-catalyzed reactions, the rate-limiting step is the slowest step.
  - Inhibiting or enhancing this specific step has the greatest impact on the final product formation.
  - Cellular or biochemical regulation is often directed at the enzyme catalyzing the rate-limiting step, providing an efficient and rapid way to control pathway output.

Chapter 6: Energy, Enzymes, and Metabolism - A Comprehensive Study Guide

Chapter 6: Energy, Enzymes, and Metabolism - A Comprehensive Study Guide