Unit 5: Enzymes and Metabolism

Enzyme Classification, Mechanism, and Kinetics

1.1 Classification and Nomenclature of Enzymes

Enzymes are biological catalysts that significantly increase the rate of biochemical reactions without being consumed in the process. They are typically proteins, highly specific for their substrates, and function under mild physiological conditions.

Enzyme Classification

The International Union of Biochemistry and Molecular Biology (IUBMB) classifies enzymes into six major classes based on the type of reaction they catalyze. Each enzyme is assigned an EC (Enzyme Commission) number, which is a numerical classification scheme.

Enzyme Nomenclature

Enzymes have both common names and systematic names (EC numbers).

• Common Name: Often ends with "-ase" and typically indicates the substrate and/or the type of reaction catalyzed (e.g., urease, alcohol dehydrogenase). These names are convenient but can sometimes be ambiguous.
• Systematic Name (EC Number): A more precise and unambiguous nomenclature based on the IUBMB classification. It consists of four numbers, for example, EC 1.1.1.27 for lactate dehydrogenase, where each number denotes a specific level of classification within the hierarchy.

1.2 Mechanism of Enzyme Action

Enzymes facilitate biochemical reactions primarily by lowering the activation energy (Ea) required for the reaction to proceed. They achieve this by providing an alternative reaction pathway.

• Active Site: A specific region on the enzyme, usually a pocket or cleft, where the substrate binds. The active site is highly specific in its shape and chemical environment, ensuring that only specific substrates can bind.
• Substrate Specificity: Enzymes are highly specific, meaning each enzyme typically catalyzes only one type of reaction or acts on a limited range of substrates. This specificity is crucial for maintaining metabolic control.
• Models of Enzyme-Substrate Interaction:
  • Lock-and-Key Model: Proposed by Emil Fischer, this model suggests that the active site has a rigid shape perfectly complementary to the substrate, like a lock and its specific key. While useful for initial understanding, it's an oversimplification.
  • Induced Fit Model: A more widely accepted model proposed by Daniel Koshland. It suggests that the active site is flexible and undergoes a conformational change upon substrate binding, optimizing the fit and enhancing catalysis. This induced fit brings catalytic groups into proper alignment.
• Role of Cofactors and Coenzymes: Many enzymes require non-protein components for their activity.
  • Cofactors: General term for non-protein chemical compounds that are bound to an enzyme and are required for the enzyme's biological activity. Can be inorganic ions (e.g., Mg²⁺, Zn²⁺, Fe²⁺) or organic molecules.
  • Coenzymes: Organic cofactors, often derived from vitamins (e.g., NAD⁺ from niacin, FAD from riboflavin, CoA from pantothenic acid). They often carry specific functional groups or electrons.
  • Prosthetic Groups: Coenzymes or metal ions that are very tightly (often covalently) bound to the enzyme.

Enzyme Mechanism Summary: Enzymes lower the activation energy by stabilizing the transition state, orienting substrates correctly, straining substrate bonds, or providing a favorable microenvironment.

1.3 Enzyme Kinetics

Enzyme kinetics is the study of the rates of enzyme-catalyzed reactions and the factors that influence these rates. It provides insights into the catalytic mechanism, specificity, and regulation of enzymes.

Key Terms and Concepts

• Reaction Rate (Velocity, V): The speed at which a product is formed or a substrate is consumed.
• Initial Velocity (V₀): The reaction rate measured at the very beginning of the reaction, before product accumulation becomes significant and substrate concentration has decreased considerably. This is often used in kinetic studies to avoid complications from reverse reactions or product inhibition.

Michaelis-Menten Kinetics

The Michaelis-Menten model describes the relationship between reaction velocity and substrate concentration for many enzymes.

Michaelis-Menten Equation:V₀ = (Vmax * [S]) / (Km + [S])

Where:

• V₀ = initial reaction velocity
• Vmax = maximum reaction velocity when the enzyme is saturated with substrate
• [S] = substrate concentration
• Km = Michaelis constant

• Vmax (Maximum Velocity): Represents the maximum rate of reaction when all enzyme active sites are saturated with substrate. It reflects the enzyme's catalytic efficiency when operating at full capacity.
• Km (Michaelis Constant):
  • The substrate concentration at which the reaction velocity is half of Vmax (V₀ = Vmax / 2).
  • It is an inverse measure of the enzyme's affinity for its substrate. A low Km indicates high affinity (enzyme reaches Vmax/2 at low [S]). A high Km indicates low affinity (requires high [S] to reach Vmax/2).
• Lineweaver-Burk Plot (Double Reciprocal Plot): A linear transformation of the Michaelis-Menten equation.

  Lineweaver-Burk Equation:1/V₀ = (Km/Vmax)(1/[S]) + 1/Vmax

  This plot is useful for determining Vmax and Km more accurately and for analyzing enzyme inhibition types. The y-intercept is 1/Vmax, and the x-intercept is -1/Km.

Factors Affecting Enzyme Activity

• Substrate Concentration: As [S] increases, V₀ increases until Vmax is reached (enzyme saturation).
• Enzyme Concentration: V₀ is directly proportional to enzyme concentration, assuming [S] is abundant.
• Temperature:
  • Increasing temperature generally increases reaction rate (due to increased kinetic energy) up to an optimal temperature.
  • Beyond the optimal temperature, enzymes begin to denature, losing their 3D structure and activity.
• pH:
  • Each enzyme has an optimal pH at which it exhibits maximum activity.
  • Deviations from the optimal pH can alter the ionization state of amino acid residues in the active site and disrupt enzyme structure, leading to denaturation and loss of activity.

Enzyme Inhibition

Inhibitors are molecules that decrease enzyme activity. They are crucial for metabolic regulation and drug design.

Carbohydrate Metabolism Pathways

2.1 Glycolysis

Glycolysis, meaning "sugar splitting," is a metabolic pathway that breaks down glucose into two molecules of pyruvate. It is the first step in carbohydrate metabolism and occurs in the cytoplasm of virtually all cells.

• Location: Cytosol
• Overall Reaction:

  Glucose + 2 NAD⁺ + 2 ADP + 2 Pi → 2 Pyruvate + 2 NADH + 2 H⁺ + 2 ATP + 2 H₂O

• Phases:
  1. Energy-Investment Phase: Glucose is phosphorylated twice, consuming 2 ATP molecules, to form fructose-1,6-bisphosphate. This molecule is then split into two 3-carbon molecules (glyceraldehyde-3-phosphate).
  2. Energy-Payoff Phase: The two 3-carbon molecules are converted to pyruvate. During this phase, 4 ATP molecules are produced via substrate-level phosphorylation, and 2 NADH molecules are generated.
• Net Products per Glucose Molecule:
  • 2 ATP (net gain, 4 produced - 2 consumed)
  • 2 NADH
  • 2 Pyruvate
• Important Observations:
  • Glycolysis can occur both in the presence (aerobic) and absence (anaerobic) of oxygen.
  • Under aerobic conditions, pyruvate proceeds to the Krebs cycle.
  • Under anaerobic conditions, pyruvate is converted to lactate (in animals) or ethanol (in yeast) through fermentation to regenerate NAD⁺.
  • Key regulatory enzymes include Hexokinase, Phosphofructokinase-1 (PFK-1), and Pyruvate Kinase. PFK-1 is the most important regulatory step.

2.2 Krebs Cycle (Citric Acid Cycle / TCA Cycle)

The Krebs cycle is a central metabolic pathway that completes the oxidation of acetyl-CoA, derived from carbohydrates, fats, and proteins, into carbon dioxide. It is a major source of electron carriers (NADH and FADH₂) for the electron transport chain.

• Location: Mitochondrial matrix
• Precursor: Acetyl-CoA (formed from pyruvate oxidation, fatty acid beta-oxidation, and amino acid catabolism).
• Overview: Acetyl-CoA condenses with oxaloacetate to form citrate. Through a series of eight steps, citrate is systematically oxidized, regenerating oxaloacetate to continue the cycle.
• Products per Acetyl-CoA Molecule:
  • 3 NADH
  • 1 FADH₂
  • 1 GTP (which is readily converted to ATP)
  • 2 CO₂ (released as waste product)
• Important Observations:
  • The Krebs cycle is an aerobic pathway, as it requires oxygen for the regeneration of NAD⁺ and FAD via the electron transport chain.
  • It is a crucial amphibolic pathway, meaning it participates in both catabolic (breakdown) and anabolic (synthesis) processes. Many intermediates serve as precursors for biosynthesis (e.g., amino acids, heme).

2.3 Pentose Phosphate Pathway (PPP) / Hexose Monophosphate Shunt

The Pentose Phosphate Pathway is an alternative route for glucose oxidation, distinct from glycolysis. Its primary roles are to produce NADPH and the precursor for nucleotide biosynthesis, ribose-5-phosphate.

• Location: Cytosol
• Phases:
  1. Oxidative Phase: Irreversible reactions that produce NADPH and ribulose-5-phosphate (from glucose-6-phosphate). The key enzyme is Glucose-6-phosphate dehydrogenase (G6PD).
  2. Non-oxidative Phase: Reversible reactions that interconvert various phosphorylated sugars, ultimately leading to the production of ribose-5-phosphate and intermediates that can re-enter glycolysis (fructose-6-phosphate and glyceraldehyde-3-phosphate).
• Main Products and Roles:
  • NADPH:
    • Used in reductive biosynthesis (e.g., fatty acid synthesis, steroid synthesis).
    • Crucial for antioxidant defense, protecting cells from reactive oxygen species by maintaining reduced glutathione.
  • Ribose-5-Phosphate: A precursor for the synthesis of nucleotides (DNA, RNA) and coenzymes (ATP, NAD⁺, FAD, CoA).
• Important Observations:
  • Cells with high demands for reductive biosynthesis (e.g., liver, adipose tissue) or protection against oxidative stress (e.g., red blood cells) have high PPP activity.
  • Deficiency in Glucose-6-phosphate dehydrogenase (G6PD) can lead to hemolytic anemia, as red blood cells lose their ability to combat oxidative stress.

2.4 Electron Transport Chain (ETC)

The Electron Transport Chain is the final stage of aerobic respiration, where the energy stored in NADH and FADH₂ (produced during glycolysis, pyruvate oxidation, and the Krebs cycle) is used to generate a proton gradient, which then drives ATP synthesis.

• Location: Inner mitochondrial membrane
• Components: A series of protein complexes (Complex I, II, III, IV) and mobile electron carriers (Ubiquinone/Coenzyme Q, Cytochrome c).
• Process:
  1. Electron Flow: Electrons from NADH enter at Complex I, and electrons from FADH₂ enter at Complex II. Electrons are then passed sequentially through Complexes III and IV to the final electron acceptor, oxygen.
  2. Proton Pumping: As electrons move through Complexes I, III, and IV, energy is released, which is used to pump protons (H⁺) from the mitochondrial matrix into the intermembrane space. This creates an electrochemical proton gradient (proton-motive force) across the inner mitochondrial membrane.
  3. Chemiosmosis and ATP Synthesis: Protons flow back into the mitochondrial matrix through a specialized protein complex called ATP synthase (Complex V). The energy released by this proton flow (proton-motive force) drives the synthesis of ATP from ADP and Pi, a process called oxidative phosphorylation.
• Final Electron Acceptor: Molecular oxygen (O₂), which accepts electrons and protons to form water (H₂O).
• ATP Yield:
  • Approximately 2.5 ATP per NADH molecule.
  • Approximately 1.5 ATP per FADH₂ molecule.
  • The total ATP yield from the complete oxidation of one glucose molecule is typically around 30-32 ATP, mainly from ETC.
• Important Observations:
  • The ETC is strictly aerobic because oxygen is essential as the final electron acceptor.
  • Uncouplers (e.g., DNP) can dissipate the proton gradient, leading to heat production instead of ATP synthesis.
  • Inhibitors (e.g., cyanide, carbon monoxide) can block electron flow, halting ATP synthesis and leading to cell death.

Lipid and Amino Acid Metabolism

3.1 β-oxidation of Fatty Acid

Beta-oxidation is the primary metabolic pathway for the catabolism of fatty acids, breaking them down into acetyl-CoA molecules, which can then enter the Krebs cycle for further oxidation to produce ATP.

• Location: Mitochondrial matrix (after initial activation and transport).
• Steps for Entry into Mitochondria:
  1. Activation: Fatty acids are activated in the cytosol by attaching to Coenzyme A (CoA), forming fatty acyl-CoA, consuming 1 ATP (converted to AMP + 2 Pi, equivalent to 2 ATP).
  2. Transport: Long-chain fatty acyl-CoAs are transported into the mitochondrial matrix via the carnitine shuttle system.
• The β-Oxidation Cycle (for saturated fatty acids): Each cycle of β-oxidation removes two carbons from the fatty acyl-CoA, producing one acetyl-CoA, one NADH, and one FADH₂. The cycle consists of four enzymatic reactions:
  1. Dehydrogenation (FADH₂ production): Acyl-CoA dehydrogenase catalyzes the removal of two hydrogens, forming a double bond and reducing FAD to FADH₂.
  2. Hydration: Enoyl-CoA hydratase adds water across the double bond.
  3. Dehydrogenation (NADH production): Hydroxyacyl-CoA dehydrogenase oxidizes the hydroxyl group, reducing NAD⁺ to NADH.
  4. Thiolysis (Acetyl-CoA release): Thiolase cleaves the bond, releasing one acetyl-CoA and a fatty acyl-CoA that is two carbons shorter, ready for the next cycle.
• Products per Cycle:
  • 1 Acetyl-CoA
  • 1 NADH
  • 1 FADH₂
• Important Observations:
  • Beta-oxidation continues until the entire fatty acid chain is converted into acetyl-CoA units.
  • For a fatty acid with 'n' carbons, it undergoes (n/2 - 1) cycles of beta-oxidation and produces (n/2) acetyl-CoA molecules. For example, a 16-carbon palmitate yields 8 acetyl-CoA, 7 NADH, and 7 FADH₂.
  • The acetyl-CoA then enters the Krebs cycle, and NADH/FADH₂ enter the electron transport chain for ATP production. Fatty acid oxidation is a highly efficient energy-generating pathway.

3.2 Transamination

Transamination is a crucial reaction in amino acid metabolism, involving the transfer of an amino group (–NH₂) from an amino acid to an α-keto acid. This process interconverts amino acids and α-keto acids, linking amino acid metabolism with carbohydrate and lipid metabolism.

• Definition: The enzymatic transfer of an α-amino group from an α-amino acid to the α-keto carbon atom of an α-keto acid, forming a new α-amino acid and a new α-keto acid.
• Enzymes: Catalyzed by enzymes called aminotransferases or transaminases.
  • Alanine aminotransferase (ALT): Transfers the amino group from alanine to α-ketoglutarate, forming pyruvate and glutamate.
  • Aspartate aminotransferase (AST): Transfers the amino group from aspartate to α-ketoglutarate, forming oxaloacetate and glutamate.

  Both ALT and AST are important clinical markers for liver damage.

• Cofactor: All aminotransferases require pyridoxal phosphate (PLP), a derivative of vitamin B₆, as a coenzyme. PLP acts as an intermediate amino group carrier.
• Overall Reaction (General):

  Amino Acid₁ + α-Keto Acid₂ ⇌ α-Keto Acid₁ + Amino Acid₂

• Importance:
  • Synthesis of non-essential amino acids.
  • Collection of amino groups from various amino acids into glutamate, which can then undergo oxidative deamination.
  • Link between amino acid metabolism and carbohydrate metabolism (e.g., pyruvate and oxaloacetate are Krebs cycle intermediates).

3.3 Deamination

Deamination is the process of removing an amino group from an amino acid, typically releasing it as ammonia (NH₃). This is a critical step in the catabolism of amino acids, especially when they are used for energy production or converted to other molecules.

• Definition: The removal of an amino group from an organic compound, typically an amino acid, resulting in the production of ammonia and an α-keto acid.
• Types of Deamination:
  • Oxidative Deamination: The most common form, primarily catalyzed by glutamate dehydrogenase, which removes the amino group from glutamate to form α-ketoglutarate and free ammonia. This reaction can use either NAD⁺ or NADP⁺ as an electron acceptor.

    Glutamate + NAD⁺/NADP⁺ + H₂O → α-Ketoglutarate + NH₃ + NADH/NADPH + H⁺

  • Non-oxidative Deamination: Less common, involving other mechanisms like hydrolytic or eliminative deamination (e.g., by serine/threonine dehydratase).
• Importance:
  • Removal of excess nitrogen from the body.
  • Production of ammonia, which is then channeled into the urea cycle for detoxification.
  • Formation of α-keto acids, which can be oxidized for energy (enter Krebs cycle) or converted to glucose or fatty acids.

3.4 Urea Cycle

The Urea Cycle (also known as the Ornithine Cycle) is the metabolic pathway that converts highly toxic ammonia (NH₃), primarily derived from amino acid deamination, into less toxic urea. Urea is then transported in the blood to the kidneys for excretion in urine.

• Location: Occurs in the liver, with enzymes located in both the mitochondrial matrix and the cytosol.
• Inputs:
  • Ammonia (NH₃) from deamination, primarily as free NH₃ or carbamoyl phosphate.
  • Carbon dioxide (CO₂) as bicarbonate (HCO₃⁻).
  • Aspartate, which provides the second nitrogen atom.
• Overall Reaction:

  2 NH₃ + CO₂ + 3 ATP + H₂O → Urea + 2 ADP + AMP + 4 Pi + Fumarate

  Note: 3 ATP molecules are consumed, but 4 high-energy phosphate bonds are cleaved (2 ATP to 2 ADP, and 1 ATP to AMP + 2 Pi, where the pyrophosphate is hydrolyzed).

• Key Steps and Intermediates:
  1. Formation of Carbamoyl Phosphate: In the mitochondrial matrix, ammonia (NH₃) and bicarbonate (HCO₃⁻) combine to form carbamoyl phosphate. This ATP-dependent reaction is catalyzed by Carbamoyl Phosphate Synthetase I (CPSI), the rate-limiting step.
  2. Formation of Citrulline: Carbamoyl phosphate transfers its carbamoyl group to ornithine, forming citrulline. Catalyzed by Ornithine Transcarbamoylase (OTC). Citrulline then moves from the mitochondria to the cytosol.
  3. Formation of Argininosuccinate: In the cytosol, citrulline condenses with aspartate (providing the second nitrogen) to form argininosuccinate. This step is catalyzed by Argininosuccinate Synthetase and consumes ATP.
  4. Cleavage of Argininosuccinate: Argininosuccinate is cleaved by Argininosuccinate Lyase, yielding fumarate and arginine. Fumarate can re-enter the Krebs cycle.
  5. Formation of Urea: Arginine is hydrolyzed by Arginase, releasing urea and regenerating ornithine. Ornithine then re-enters the mitochondrial matrix to restart the cycle.
• Important Observations:
  • The urea cycle is essential for detoxifying ammonia, which is highly toxic to the central nervous system.
  • Defects in urea cycle enzymes can lead to hyperammonemia, a life-threatening condition.
  • The cycle links to the Krebs cycle via fumarate and aspartate.