Chapter 6: Energy, Enzymes, and Metabolism

This chapter introduces the fundamental concepts of energy, enzymes, and metabolism, which are crucial for understanding how living cells function. It begins by illustrating the role of enzymes through the example of common medications like aspirin and ibuprofen, then delves into the principles of energy and chemical reactions, the catalytic action of enzymes and ribozymes, and the intricate regulation of metabolic pathways.

Introduction to Enzymes and Metabolism

Aspirin and Ibuprofen as Enzyme Inhibitors:
- Humans have used willow tree bark (containing salicylic acid) for pain relief for over 2,000 years.
- Modern aspirin is acetylsalicylic acid, a derivative gentler on the stomach.
- These drugs work by inhibiting a specific enzyme called 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 act as critical catalysts, significantly speeding up thousands of different chemical reactions within cells.
- A chemical reaction is a process where one or more substances are transformed into other substances. This can involve:
  - 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 sustain life.
Metabolism Defined:
- Metabolism is the sum total of all chemical reactions occurring within an organism.
- It also refers to specific sets of chemical reactions at the cellular level, such as sugar metabolism or fat metabolism.
- Most metabolic processes involve either the breakdown (catabolism) or synthesis (anabolism) of organic molecules.
- Organic molecules are vital for maintaining cell structure, providing building blocks, and storing chemical energy in their bonds to drive cellular processes.
Chapter Overview:
- General discussion of energy and chemical reactions.
- Factors controlling the direction and rate of chemical reactions, with a focus on enzymes.
- Metabolism at the cellular level, including coordinated metabolic pathways.
- Regulation of metabolic pathways and the recycling of organic molecules.

6.1 Energy and Chemical Reactions

The fate of a chemical reaction in a living cell is governed by two primary factors: its direction and its rate. Cells utilize energy intermediate molecules, such as ATP, to control the direction of these reactions.

Factors Governing Chemical Reactions

Direction: Whether reactants are converted to products or vice versa.
Rate: How quickly the reaction proceeds.
Generalized Chemical Reaction:
- aA + bB ⇌ cC + dD
- A and B are reactants, C and D are products.
- a, b, c, d represent the number of moles.
- The double arrow (⇌) indicates reversibility.
- The direction depends on energy considerations and the concentrations of reactants and products.

Energy Exists in Different Forms

Definition of Energy: The ability to cause change or do work.
Two General Forms of Energy:
- 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's potential energy is based on its position relative to the nucleus and other electrons. Electrons in outer shells have higher potential energy; dropping to an inner shell converts potential energy 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, which is released during breakdown to produce energy intermediates like ATP.
Thermodynamics: The study of energy interconversions.
- 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.
  - Entropy: A measure of the randomness of molecules in a system. 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

Usable vs. Unusable Energy:
- Cells require usable energy for processes like chemical reactions, cellular movements (e.g., muscle contraction), and maintaining cell organization.
- Total energy = Usable energy + Unusable energy.
- Unusable energy arises from entropy, which cannot be harnessed usefully.
Enthalpy, Free Energy, and Entropy Relationship:
- 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 (disorder).
- The relationship is expressed as: H = G + TS, where T is the absolute temperature in kelvins (K).
- Rearranged for free energy: G = H – TS.
Spontaneity of Reactions:
- A critical question in biology is whether a process occurs spontaneously (e.g., glucose breakdown).
- A spontaneous reaction does not imply a fast reaction; its rate can be very slow (e.g., sugar breakdown in a bowl).
Free-Energy Change (ΔG):
- The key to evaluating spontaneity is the free-energy change (ΔG) during a reaction: ΔG = ΔH – TΔS.
- Exergonic Reactions:
  - Occur when ΔG < 0 (negative free-energy change).
  - Products have less free energy than reactants.
  - Free energy is released during product formation.
  - These reactions are spontaneous.
- Endergonic Reactions:
  - Occur when ΔG > 0 (positive free-energy change).
  - Require the addition of free energy.
  - These reactions are not spontaneous.
- A negative ΔG favors product formation, while a positive ΔG favors reactant formation.
- Example: ATP Hydrolysis
  - 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.
  - For 1 mole of ATP to ADP + Pi, ΔG = –7.3 kcal/mol.
  - This negative value indicates that product formation is strongly favored, and energy is liberated.

Cells Use ATP to Drive Endergonic Reactions

Coupling 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 of Coupled Reaction (Glucose Phosphorylation):
- Endergonic reaction: Glucose + Phosphate²⁻ → Glucose-6-phosphate²⁻ + H₂O (ΔG = +3.3 kcal/mol) - not spontaneous alone.
- Exergonic reaction: ATP⁴⁻ + H₂O → ADP²⁻ + Pi²⁻ (ΔG = −7.3 kcal/mol).
- Coupled reaction: Glucose + ATP⁴⁻ → Glucose-6-phosphate²⁻ + ADP²⁻ (ΔG = −4.0 kcal/mol).
- In the coupled reaction, a phosphate is directly transferred from ATP to glucose, a process called phosphorylation.
- The net negative ΔG makes the coupled reaction spontaneous.
ATP Recycling:
- Human cells use millions of ATP molecules per second for endergonic processes.
- The breakdown of food molecules (exergonic) releases energy to synthesize more ATP from ADP and phosphate (endergonic).
- This recycling is incredibly rapid: an average person hydrolyzes about 100 pounds of ATP daily, with each ATP molecule undergoing approximately 10,000 cycles of hydrolysis and regeneration.

Genomes Encode Many Proteins That Use ATP as a Source of Energy

ATP-Binding Sites:
- Researchers have identified specific amino acid sequences within proteins that function as ATP-binding sites.
- This allows prediction of whether a newly discovered protein uses ATP.
Proteome Analysis:
- By analyzing entire genome sequences, scientists can identify genes encoding proteins with ATP-binding sites.
- Proteomes are all the proteins a given cell or organism makes.
- Studies show that, on average, over 20% of all proteins bind ATP, though this is likely an underestimate.
- In humans, with an estimated 22,000 protein-coding genes, at least 4,400 encode ATP-using proteins.
- This highlights the immense importance of ATP as an energy source for living cells.

6.2 Enzymes and Ribozymes

While thermodynamics dictates the direction of a chemical reaction, catalysts are essential to achieve a rapid reaction rate in living cells. Enzymes, primarily proteins, are the most common biological catalysts, though some RNA molecules (ribozymes) also possess catalytic activity.

Enzymes Increase the Rates of Chemical Reactions

Catalyst Definition: An agent that speeds up a chemical reaction's rate without being permanently changed or consumed.
Enzymes: The most common catalysts in living cells, composed of proteins. The term "enzyme" was coined by Wilhelm Kuhne in 1876 after discovering trypsin.
Ribozymes: Some biological catalysts are RNA molecules.
Rate Enhancement:
- Even spontaneous (exergonic) reactions can be very slow without a catalyst (e.g., gasoline breakdown).
- Enzyme-catalyzed reactions are typically millions of times faster than uncatalyzed reactions.
- Example: The enzyme catalase speeds up the breakdown of hydrogen peroxide (H₂O₂) into water and oxygen by 10¹⁵-fold.
Mechanism of Catalysis: Lowering Activation Energy:
- Chemical reactions involve breaking and forming bonds, which requires straining existing bonds or precise positioning of molecules.
- An initial input of energy, called activation energy (E_A), is required to overcome electron repulsion and allow molecules to get close enough for bond rearrangement.
- This energy allows reactants to reach a transition state, where original bonds are stretched to their limit, after which products can readily form.
- The activation energy barrier is why many reactions are slow.
- Enzymes lower the activation energy, enabling a small amount of available heat to push reactants to the transition state.
- Two common ways enzymes lower E_A:
  - Straining reactants: When reactant molecules bind to an enzyme, their bonds can be strained, making it easier to achieve the transition state.
  - Positioning reactants close together: For reactions with multiple reactants, the enzyme provides a site where they 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 takes place.
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: The temporary complex formed when an enzyme binds to its substrate(s).
High Specificity:
- Enzymes typically bind their substrates with a high degree of specificity (e.g., hexokinase recognizes glucose but not fructose or galactose well).
- Lock and Key Model (Emil Fischer, 1894): An early model suggesting that enzyme-substrate recognition is like a specific key (substrate) fitting into a specific lock (active site).
Induced Fit (Daniel Koshland, 1958):
- Beyond the lock-and-key model, research showed that enzyme-substrate interaction involves conformational changes (movements) in the enzyme itself.
- These changes cause the substrates to bind more tightly to the enzyme, a phenomenon called induced fit.
- Induced fit is crucial for lowering activation energy and occurs before the enzyme catalyzes the conversion of reactants to products.

Enzyme Function Is Influenced by the Substrate Concentration and by Inhibitors

Enzyme Affinity:
- The degree of attraction between an enzyme and its substrate(s).
- High affinity enzymes bind substrates even at low concentrations.
- Low affinity enzymes require higher substrate concentrations for complex formation.
Substrate Concentration and Reaction Velocity:
- As substrate concentration increases, the reaction velocity (rate of product production) increases.
- Eventually, the velocity reaches a plateau because nearly all active sites are occupied by substrate. At this point, the enzyme is saturated.
- Vmax: The maximal rate of the chemical reaction when the enzyme is saturated with substrate.
- KM (Michaelis Constant): The substrate concentration at which the reaction velocity is half its maximal value (Vmax/2).
  - Named after Leonor Michaelis.
  - KM measures the substrate concentration required for a reaction to occur.
  - High KM indicates low affinity (weaker binding), requiring higher substrate concentration for a given rate.
  - Low KM indicates high affinity (stronger binding).
- The enzyme-catalyzed reaction can be viewed as two steps: E + S ⇌ ES → E + P (Enzyme + Substrate ⇌ Enzyme-Substrate Complex → Enzyme + Product).
- If product formation is much slower than substrate release, KM is inversely related to enzyme-substrate affinity.
Enzyme Inhibitors: Molecules that decrease enzyme function.
- Reversible Inhibitors: Bind noncovalently and modulate enzyme function, allowing the enzyme to be reused.
  - Competitive Inhibitors:
    - Bind noncovalently to the enzyme's active site.
    - Compete with the substrate for binding.
    - Often mimic the substrate's structure.
    - Increase the KM for the substrate, meaning a higher substrate concentration is needed to achieve the same reaction rate.
    - 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 without affecting the KM.
    - Inhibit enzyme function by causing a conformational change that affects the active site's efficiency.
- Irreversible Inhibitors: Usually bind covalently to an enzyme, permanently inactivating its function.
  - Example: Diisopropyl phosphorofluoridate (DIFP), a nerve gas, covalently reacts with acetylcholinesterase, an enzyme crucial for neuron function.
  - Irreversible inhibition is not a common cellular control mechanism because it is energy-inefficient (the 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 to the enzyme surface and promote a chemical reaction.
- Coenzymes: Organic molecules that temporarily bind to an enzyme, participate in the catalyzed reaction, but are left unchanged.
Environmental Conditions:
- Temperature: Most enzymes have an optimal temperature range.
  - Human enzymes typically function best at 37°C (normal body temperature).
  - Temperatures significantly above or below optimal inhibit function.
  - Very high temperatures can denature a protein, causing it to unfold, lose its three-dimensional shape, and thus its function.
- pH: Enzyme function is highly sensitive to pH.
  - Example: Pepsin, a protease in the stomach, functions optimally at an acidic pH of ~2.0.
  - Many cytosolic enzymes function best at a more neutral pH, such as 7.2.
  - Significant deviations from optimal pH decrease enzyme function.

Feature Investigation | The Discovery of Ribozymes by Sidney Altman Revealed That RNA Molecules May Also Function as Catalysts

Prior Belief: Until the 1980s, all biological catalysts were thought to be proteins.
RNase P Discovery:
- Sidney Altman and colleagues studied ribonuclease P (RNase P), an enzyme involved in processing tRNA molecules (precursor tRNAs, ptRNAs).
- RNase P cleaves a specific covalent bond in ptRNA, releasing a 5′ fragment and shortening the molecule.
- Purification of RNase P from E. coli revealed it has two subunits: an RNA molecule (377 nucleotides) and a small protein (14 kDa). This was highly unexpected.
In Vitro Experimentation:
- Cecilia Guerrier-Takada in Altman's lab found that magnesium ion (Mg²⁺) stimulates RNase P function.
- An experiment investigated the effects of low (10 mM MgCl₂) and high (100 mM MgCl₂) magnesium concentrations on ptRNA processing.
- Samples were incubated with: no RNase P (control), the RNA subunit alone, or intact RNase P (RNA + protein subunits).
- Gel electrophoresis was used to determine if ptRNAs were cleaved into tRNA and a 5′ fragment.
Experimental Results:
- Control (no RNase P): No processing observed at low or high MgCl₂.
- Low MgCl₂, RNA subunit alone: No processing.
- Low MgCl₂, intact RNase P: Processing occurred.
- High MgCl₂, RNA subunit alone: Processing occurred! The RNA subunit by itself was sufficient to cleave ptRNA.
- This suggested that high MgCl₂ concentration helps the RNA subunit maintain a catalytically active conformation, similar to the role of the protein subunit in a living cell.
Conclusion and Impact:
- Subsequent work confirmed the RNA subunit of RNase P is a true catalyst.
- Around the same time, Thomas Cech and colleagues discovered catalytic activity in a different RNA molecule from Tetrahymena thermophila.
- The term ribozyme was coined to describe RNA molecules that catalyze chemical reactions.
- Altman and Cech received the Nobel Prize in Chemistry in 1989 for their discovery.
- Since then, many ribozymes have been found to play key catalytic roles in cells.

6.3 Overview of Metabolism

In living cells, individual chemical reactions are not isolated but are coordinated within metabolic pathways. These pathways are broadly categorized into catabolic (breakdown) and anabolic (synthesis) reactions, both of which are tightly regulated.

Catabolic Reactions Recycle Organic Building Blocks and Produce Energy Intermediates Such as ATP

Definition: Catabolic reactions break down larger molecules into smaller ones, often releasing energy (exergonic).
Two Main Uses of Catabolic Reactions:
- Recycling of Organic Building Blocks:
  - Macromolecules are broken down to recycle their constituent organic molecules.
  - These smaller molecules then serve as building blocks for new molecules and macromolecules.
  - Example: Proteins (polypeptides) are broken down into individual amino acids by enzymes called proteases when they are misfolded or no longer needed. These amino acids can then be reused.
- Breakdown of Organic Molecules to Obtain Energy:
  - Covalent bonds in organic molecules store significant energy.
  - Cells do not directly use this released energy but store it in energy intermediates, such as ATP, which then directly drive endergonic reactions.
  - Example: The breakdown of glucose into two pyruvate molecules via glycolysis (a catabolic pathway).
  - Energy released from breaking glucose bonds is harnessed to synthesize ATP.
  - Substrate-Level Phosphorylation: A mechanism where an enzyme directly transfers a phosphate group from an organic molecule (e.g., phosphoenolpyruvate) to ADP, forming ATP.
    - Phosphoenolpyruvate has an unstable phosphate bond.
    - Phosphoenolpyruvate + ADP → Pyruvate + ATP (ΔG = −7.5 kcal/mol, an exergonic reaction).
  - Chemiosmosis: Another mechanism for ATP synthesis, utilizing 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 "reduction" 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): When an atom or molecule is oxidized, the removed electron is transferred to another atom or molecule, which then becomes reduced.
  - Generalized equation: Ae⁻ + B → A (oxidized) + Be⁻ (reduced).
- Role in Energy Intermediates:
  - During the oxidation of organic molecules (e.g., glucose), removed electrons are used to produce energy intermediates like NADH.
  - Nicotinamide adenine dinucleotide (NAD⁺) is reduced to NADH.
  - Uses of NADH:
    - Its oxidation is a highly exergonic reaction that can be used to make ATP.
    - It can donate electrons to other organic molecules, energizing them for covalent bond formation, particularly in anabolic reactions.

Anabolic Reactions Require an Input of Energy to Make Larger Molecules

Definition: Also called biosynthetic reactions, they involve the synthesis of larger molecules and macromolecules from smaller precursor molecules.
Energy Requirement: Anabolic reactions are typically endergonic and require an input of energy, often supplied by energy intermediates like ATP or NADH.
Examples:
- Synthesis of macromolecules like RNA and proteins.
- Synthesis of small organic molecules such as amino acids and fats, especially if they are not available from food sources.
- These involve forming covalent linkages between precursor molecules.
- Example: Glutamate (an amino acid) is synthesized by covalently linking α-ketoglutarate (a sugar metabolism product) and ammonium (NH₄⁺), a reaction that requires NADH.

Metabolic Pathways Are Regulated in Three General Ways

Regulation of metabolic pathways is crucial to ensure that organic molecules are broken down only when needed for energy or recycling, and synthesized only when required by the cell, preventing wasteful overaccumulation.

1. Gene Regulation:
- Enzymes are proteins encoded by genes.
- Cells control metabolic pathways by regulating gene expression (turning genes on or off).
- Example: A bacterial cell will turn off genes for enzymes that break down a specific sugar if that sugar is absent, and turn them back on when it becomes available.
2. Regulation via Cell-Signaling Pathways:
- Metabolism is coordinated at the cellular level, integrating environmental signals.
- Cell-signaling pathways often activate protein kinases, enzymes that covalently attach a phosphate group to a target protein.
- Example: When frightened, humans secrete epinephrine (the fight-or-flight hormone).
  - Epinephrine binds to muscle cells, stimulating an intracellular pathway.
  - This leads to the phosphorylation of specific enzymes involved in carbohydrate metabolism.
  - These activated enzymes promote carbohydrate breakdown, providing more energy for the individual.
  - When the fright subsides, phosphatases remove the phosphate groups, restoring normal carbohydrate metabolism.
3. Biochemical Regulation:
- Involves the direct regulation of enzyme function through the noncovalent binding of molecules.
- Includes competitive and noncompetitive inhibitors (as discussed previously).
- Feedback Inhibition: A common type of biochemical regulation where the final product of a metabolic pathway inhibits an enzyme that acts early in the pathway.
  - This prevents the overaccumulation of the product.
  - It 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 a molecule (often the pathway's product) to the allosteric site causes a conformational change in the enzyme, inhibiting its catalytic function (e.g., by altering the active site).
  - Allosteric sites are frequently found in enzymes catalyzing the early, committed steps of a pathway.
  - This mechanism is a form of noncompetitive inhibition.
Regulation of the Rate-Limiting Step:
- In metabolic pathways with multiple enzyme-catalyzed steps, regulation is often directed at the rate-limiting step, which is the slowest step.
- Inhibiting or enhancing this single step has the greatest impact on the overall rate of final product formation.
- This is an efficient and rapid way to control the amount of product in a pathway, rather than regulating every enzyme.