Unit 5: Enzymes and Metabolism

Enzymes: Classification, Mechanism, and Kinetics

What are Enzymes?

Enzymes are biological catalysts, primarily proteins, that accelerate the rate of biochemical reactions without being consumed in the process. They are highly specific for their substrates and reactions.

Definition: Enzymes are globular proteins that function as biological catalysts, increasing the rate of specific biochemical reactions by lowering the activation energy.

Key Characteristics of Enzymes:

• Catalytic Power: They can increase reaction rates by factors of 10⁶ to 10¹² or more.
• Specificity: They typically catalyze only one type of reaction or act on a very limited range of substrates.
• Regulation: Their activity can be regulated by various mechanisms, allowing cells to control metabolic pathways.
• Temperature and pH Sensitivity: Most enzymes have optimal temperature and pH ranges outside which their activity decreases significantly due to denaturation.

Classification and Nomenclature of Enzymes

Enzymes are systematically named and classified by the Enzyme Commission (EC) based on the type of reaction they catalyze. Each enzyme is assigned a unique EC number consisting of four digits (e.g., EC X.Y.Z.W).

Nomenclature

• Systematic Name: Provides a clear, unambiguous description of the reaction catalyzed. Often long and complex.
• Common Name: Shorter, more convenient name often used in practice. Many end with the suffix "-ase" (e.g., amylase, lipase). Some older names like pepsin or trypsin do not follow this rule.
• EC Number: A numerical classification system. The first digit indicates the main class of the enzyme.

Major Classes of Enzymes (EC Classification)

There are six main classes of enzymes:

Mechanism of Enzyme Action

Enzymes achieve their catalytic power by providing an alternative reaction pathway with a lower activation energy (ΔG‡) compared to the uncatalyzed reaction. They do not change the overall free energy change (ΔG) or the equilibrium of the reaction.

The Active Site

The active site is a specific, three-dimensional region on the enzyme where the substrate binds and catalysis occurs. It is typically a small cleft or crevice on the enzyme surface, formed by amino acid residues that may be far apart in the primary sequence but brought together by protein folding.

Characteristics of the Active Site:

• Specificity: It has a unique shape and chemical environment that allows it to bind only specific substrates.
• Optimal Orientation: It correctly positions the substrate(s) for the reaction.
• Catalytic Residues: Contains amino acid residues that directly participate in the chemical transformation of the substrate.

Models of Enzyme-Substrate Binding

• Lock and Key Model (Emil Fischer, 1894):

  This model proposes that the active site of the enzyme has a rigid, pre-formed shape that perfectly matches the shape of its substrate, much like a specific key fits into a specific lock. While useful for explaining enzyme specificity, it does not fully account for the dynamic nature of enzymes.

• Induced Fit Model (Daniel Koshland, 1958):

  This more refined model suggests that both the enzyme and the substrate undergo slight conformational changes upon binding. The substrate induces a change in the enzyme's active site, leading to a tighter fit and optimizing the catalytic process. This dynamic interaction improves catalytic efficiency and broadens the understanding of enzyme specificity.

How Enzymes Lower Activation Energy

Enzymes facilitate reactions by:

1. Proximity and Orientation: Bringing substrates together in the correct orientation to react.
2. Strain/Distortion: Inducing strain in the substrate, making it more reactive (transition state stabilization).
3. Acid-Base Catalysis: Donating or accepting protons (H⁺) from the substrate to facilitate bond breaking or formation.
4. Covalent Catalysis: Forming transient covalent bonds with the substrate during the reaction.
5. Metal Ion Catalysis: Using metal ions to facilitate substrate binding or stabilize charged transition states.

Factors Affecting Enzyme Activity

• Temperature:
  • Enzyme activity generally increases with temperature up to an optimum.
  • Beyond the optimum, high temperatures cause denaturation (unfolding of the protein), leading to a rapid loss of activity.
• pH:
  • Each enzyme has an optimal pH range where its activity is maximal.
  • Deviations from the optimal pH alter the ionization state of amino acid residues in the active site and throughout the protein, affecting substrate binding and catalytic efficiency, eventually leading to denaturation.
• Substrate Concentration:
  • At low substrate concentrations, enzyme activity increases proportionally with substrate concentration.
  • At high substrate concentrations, the enzyme becomes saturated with substrate, and the reaction rate reaches its maximum (Vmax).
• Enzyme Concentration:
  • Assuming an excess of substrate, the reaction rate is directly proportional to the enzyme concentration. More enzyme molecules mean more active sites available to convert substrate to product.
• Inhibitors:

  Molecules that bind to an enzyme and decrease its activity.

  • Competitive Inhibition: Inhibitor resembles the substrate and binds reversibly to the active site, competing with the substrate. Can be overcome by increasing substrate concentration.
  • Non-Competitive Inhibition: Inhibitor binds to an allosteric site (different from the active site), altering the enzyme's conformation and reducing its catalytic efficiency. Cannot be overcome by increasing substrate concentration.
  • Uncompetitive Inhibition: Inhibitor binds only to the enzyme-substrate complex, preventing the conversion of substrate to product.

Enzyme Kinetics

Enzyme kinetics is the study of the rates of enzyme-catalyzed reactions. It provides insights into the mechanism of enzyme action, catalytic efficiency, and regulation.

Initial Velocity (V₀)

The initial rate of an enzyme-catalyzed reaction, measured at the beginning of the reaction when substrate concentration is highest and product concentration is negligible. This minimizes the effect of reverse reactions and product inhibition.

Michaelis-Menten Kinetics

The Michaelis-Menten model describes the relationship between reaction rate and substrate concentration for many enzymes. It assumes a simple two-step reaction:

E + S ⇌ ES → E + P

Where E = Enzyme, S = Substrate, ES = Enzyme-Substrate complex, P = Product.

Michaelis-Menten Equation:

V₀ = (V_max[S]) / (K_m + [S])

Where:

• V₀ is the initial reaction velocity.
• V_max is the maximum reaction velocity when the enzyme is saturated with substrate.
• [S] is the substrate concentration.
• K_m is the Michaelis constant.

Michaelis Constant (K_m)

Definition: The Michaelis constant (K_m) is the substrate concentration at which the reaction velocity (V₀) is half of the maximum velocity (V_max).

Significance of K_m:

• It is an inverse measure of the enzyme's affinity for its substrate. A low K_m indicates high affinity (enzyme binds tightly to substrate), while a high K_m indicates low affinity (enzyme binds weakly to substrate).
• It is a characteristic constant for a particular enzyme-substrate pair under specific conditions (pH, temperature, ionic strength).

Maximum Velocity (V_max)

Definition: The maximum velocity (V_max) is the maximum rate of reaction achieved when all enzyme active sites are saturated with substrate. At this point, the enzyme is working at its full catalytic capacity.

Significance of V_max:

• It reflects the turnover number (k_cat) of the enzyme, which is the number of substrate molecules converted to product per enzyme molecule per unit time when the enzyme is saturated.
• V_max is proportional to the total enzyme concentration.

Lineweaver-Burk Plot (Double Reciprocal Plot)

The Lineweaver-Burk plot is a graphical representation of the Michaelis-Menten equation, obtained by taking the reciprocal of both sides. It converts the hyperbolic Michaelis-Menten curve into a linear plot, making it easier to determine V_max and K_m, and to analyze enzyme inhibition types.

Lineweaver-Burk Equation:

1 / V₀ = (K_m / V_max) * (1 / [S]) + (1 / V_max)

This is in the form of y = mx + c, where:

• y-intercept = 1 / V_max
• x-intercept = -1 / K_m
• Slope = K_m / V_max

Central Metabolic Pathways

Metabolism encompasses all the chemical reactions that occur within a cell or organism to maintain life. These pathways are highly interconnected and regulated.

Glycolysis

Glycolysis is the first stage of glucose catabolism. It is a universal pathway occurring in almost all organisms, both aerobic and anaerobic.

Definition: Glycolysis is a metabolic pathway that breaks down a molecule of glucose (a 6-carbon sugar) into two molecules of pyruvate (a 3-carbon compound), generating a net gain of ATP and NADH.

• Location: Cytosol of the cell.
• Oxygen Requirement: Does not require oxygen (anaerobic process).

Overall Reaction:

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

Stages of Glycolysis:

1. Energy Investment Phase (Steps 1-5):
   • Glucose is phosphorylated twice, consuming 2 molecules of ATP.
   • The 6-carbon glucose molecule is cleaved into two 3-carbon molecules of Glyceraldehyde-3-phosphate.
2. Energy Payoff Phase (Steps 6-10):
   • The two Glyceraldehyde-3-phosphate molecules are converted to pyruvate.
   • This phase generates 4 molecules of ATP (via substrate-level phosphorylation) and 2 molecules of NADH.

Net Yield per Glucose Molecule:

• 2 ATP (Net: 4 produced - 2 consumed)
• 2 NADH
• 2 Pyruvate

Fate of Pyruvate:

• Under Aerobic Conditions: Pyruvate is transported into the mitochondria and converted to Acetyl-CoA, which then enters the Krebs Cycle.
• Under Anaerobic Conditions:
  • In animals (e.g., muscle cells during intense exercise), pyruvate is converted to Lactate (Lactic Acid Fermentation) to regenerate NAD⁺.
  • In yeast and some bacteria, pyruvate is converted to Ethanol and CO₂ (Alcoholic Fermentation) to regenerate NAD⁺.

Krebs Cycle (Citric Acid Cycle / Tricarboxylic Acid Cycle - TCA Cycle)

The Krebs cycle is a central metabolic hub that completes the oxidation of acetyl-CoA derived from carbohydrates, fatty acids, and amino acids, generating reduced coenzymes (NADH and FADH2) for the electron transport chain.

Definition: The Krebs Cycle is a series of eight enzyme-catalyzed reactions that fully oxidize the acetyl group of Acetyl-CoA to two molecules of carbon dioxide, producing ATP (or GTP), NADH, and FADH2.

• Location: Mitochondrial matrix (in eukaryotes).
• Oxygen Requirement: Directly does not use oxygen but requires oxygen for the regeneration of NAD⁺ and FAD from NADH and FADH₂ via the Electron Transport Chain. Thus, it is an aerobic process.

Link Reaction: Formation of Acetyl-CoA

Before entering the Krebs cycle, pyruvate (from glycolysis) is converted to Acetyl-CoA by the pyruvate dehydrogenase complex. This irreversible step also produces one NADH and one CO₂ per pyruvate.

2 Pyruvate + 2 NAD⁺ + 2 CoA → 2 Acetyl-CoA + 2 NADH + 2 H⁺ + 2 CO₂

Key Inputs and Outputs per Acetyl-CoA molecule:

• Input: 1 Acetyl-CoA (2 carbons)
• Outputs:
  • 2 CO₂
  • 3 NADH
  • 1 FADH₂
  • 1 GTP (which can be converted to ATP)

Since one glucose molecule yields two pyruvate molecules, and thus two acetyl-CoA molecules, the total yield from one glucose molecule through the Krebs cycle is double the above outputs.

Role in Metabolism:

• Energy Production: Major source of NADH and FADH₂ for the ETC.
• Biosynthesis: Many cycle intermediates serve as precursors for the synthesis of amino acids, glucose (gluconeogenesis), fatty acids, and porphyrins. This amphibolic nature makes it a crucial central pathway.

Pentose Phosphate Pathway (PPP / Hexose Monophosphate Shunt)

The Pentose Phosphate Pathway is an alternative route for glucose-6-phosphate metabolism, running parallel to glycolysis.

Definition: The Pentose Phosphate Pathway is a metabolic pathway that generates NADPH and the 5-carbon sugar ribose-5-phosphate, which are crucial for reductive biosynthesis and nucleotide synthesis, respectively.

• Location: Cytosol of the cell.
• Oxygen Requirement: Does not directly consume or produce oxygen.

Main Functions:

1. Production of NADPH:
   • NADPH is a crucial reducing agent for anabolic (biosynthetic) reactions, such as fatty acid synthesis, cholesterol synthesis, and steroid hormone synthesis.
   • It is also vital for protecting cells from oxidative stress by maintaining reduced glutathione, which is involved in detoxifying reactive oxygen species.
2. Production of Ribose-5-Phosphate:
   • Ribose-5-phosphate is a direct precursor for the synthesis of nucleotides (DNA, RNA) and coenzymes (ATP, NADH, FADH2, CoA).

Phases of PPP:

• Oxidative Phase (Irreversible):
  • Glucose-6-phosphate is oxidized, producing NADPH and ribulose-5-phosphate.
  • This phase is the primary source of NADPH.
• Non-Oxidative Phase (Reversible):
  • Interconverts various 3, 4, 5, 6, and 7-carbon sugars.
  • Can produce ribose-5-phosphate from ribulose-5-phosphate and convert excess 5-carbon sugars into intermediates of glycolysis (fructose-6-phosphate and glyceraldehyde-3-phosphate), linking PPP to glycolysis.

Electron Transport Chain (ETC) and Oxidative Phosphorylation

The Electron Transport Chain (ETC) and Oxidative Phosphorylation are the final stages of aerobic respiration, responsible for the vast majority of ATP synthesis.

Definition: The Electron Transport Chain is a series of protein complexes and electron carriers embedded in the inner mitochondrial membrane that transfer electrons from NADH and FADH2 to molecular oxygen, creating a proton gradient. Oxidative phosphorylation is the process by which this proton gradient is used by ATP synthase to generate ATP.

• Location: Inner mitochondrial membrane (in eukaryotes).
• Oxygen Requirement: Requires oxygen as the final electron acceptor.

Components of the ETC:

The ETC consists of four major protein complexes (Complex I, II, III, IV) and two mobile electron carriers (Ubiquinone/Coenzyme Q and Cytochrome c).

1. Complex I (NADH Dehydrogenase): Accepts electrons from NADH, pumps protons.
2. Complex II (Succinate Dehydrogenase): Accepts electrons from FADH₂ (from Krebs cycle), does NOT pump protons.
3. Ubiquinone (CoQ): Lipid-soluble carrier, transports electrons from Complex I and II to Complex III.
4. Complex III (Cytochrome bc1 complex): Accepts electrons from CoQ, pumps protons.
5. Cytochrome c: Water-soluble carrier, transports electrons from Complex III to Complex IV.
6. Complex IV (Cytochrome c Oxidase): Accepts electrons from Cytochrome c, transfers them to molecular oxygen (O₂), forming water (H₂O), and pumps protons.

Mechanism of Oxidative Phosphorylation (Chemiosmotic Theory):

1. Electron Transport: NADH and FADH₂ donate their high-energy electrons to the ETC complexes. These electrons are passed sequentially from one carrier to the next, releasing energy at each step.
2. Proton Pumping: The energy released during electron transport is used by Complexes I, III, and IV 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. ATP Synthesis (Chemiosmosis): The accumulated protons flow back into the mitochondrial matrix down their concentration gradient through a specialized enzyme complex called ATP Synthase (Complex V or F₀F₁ ATPase). The energy of this proton flow drives the synthesis of ATP from ADP and inorganic phosphate (P_i).

Overall Equation for Oxidative Phosphorylation:

ADP + P_i + Energy (from proton gradient) → ATP + H₂O

Overall ATP Yield (Approximate):

Complete oxidation of one glucose molecule yields approximately 30-32 ATP molecules, with the majority produced via oxidative phosphorylation.

• Each NADH typically yields about 2.5 ATP.
• Each FADH₂ typically yields about 1.5 ATP.

Lipid and Amino Acid Metabolism

This section explores how the body processes fats and proteins for energy and other vital functions.

β-oxidation of Fatty Acids

β-oxidation is the primary pathway for the catabolism of fatty acids, breaking them down into acetyl-CoA units for energy production.

Definition: β-oxidation is a metabolic process that involves the sequential removal of two-carbon units (as acetyl-CoA) from the carboxyl end of a fatty acyl-CoA molecule, generating FADH2 and NADH with each cycle.

• Location: Mitochondrial matrix (for long-chain fatty acids). Very long-chain fatty acids undergo initial β-oxidation in peroxisomes.
• Purpose: To generate Acetyl-CoA, NADH, and FADH₂ from fatty acids for entry into the Krebs cycle and ETC.

Steps of β-oxidation:

1. Activation of Fatty Acid:

   Fatty acids are first activated in the cytosol by attaching to Coenzyme A (CoA) to form a fatty acyl-CoA, a reaction catalyzed by fatty acyl-CoA synthetase (also called thiokinase), consuming 2 ATP equivalents.

   Fatty Acid + CoA + ATP → Fatty Acyl-CoA + AMP + 2 P_i

2. Transport into Mitochondria:

   Long-chain fatty acyl-CoAs are transported into the mitochondrial matrix via the carnitine shuttle system. Short and medium-chain fatty acids can diffuse freely.

3. The β-oxidation Cycle (Four Reactions):

   Once inside the matrix, the fatty acyl-CoA undergoes a recurring sequence of four enzymatic reactions:

   1. Dehydrogenation 1: FAD-dependent oxidation (catalyzed by acyl-CoA dehydrogenase), forming a double bond and producing 1 FADH₂.
   2. Hydration: Addition of water across the double bond (catalyzed by enoyl-CoA hydratase).
   3. Dehydrogenation 2: NAD⁺-dependent oxidation (catalyzed by β-hydroxyacyl-CoA dehydrogenase), producing 1 NADH + H⁺.
   4. Thiolysis: Cleavage of the β-ketoacyl-CoA by CoA-SH (catalyzed by thiolase), releasing one molecule of Acetyl-CoA and a fatty acyl-CoA that is two carbons shorter.

   This shorter fatty acyl-CoA then re-enters the cycle until the entire fatty acid chain is broken down into acetyl-CoA units.

Yield per Cycle:

• 1 Acetyl-CoA
• 1 FADH₂
• 1 NADH

These products then feed into the Krebs Cycle (Acetyl-CoA) and Electron Transport Chain (NADH, FADH₂) for extensive ATP generation. Fatty acids are a highly efficient source of energy.

Transamination

Transamination is a crucial reaction in amino acid metabolism, involving the interconversion of amino acids and alpha-keto acids.

Definition: Transamination is the biochemical process involving the transfer of an amino group (-NH₂) from an α-amino acid to an α-keto acid, typically resulting in the formation of a new amino acid and a new α-keto acid.

• Location: Primarily in the liver and kidneys, but occurs in most tissues.
• Enzymes: Catalyzed by enzymes called aminotransferases (also known as transaminases).
• Cofactor: Requires pyridoxal phosphate (PLP), a derivative of vitamin B₆, as a prosthetic group.

General Reaction:

Amino Acid 1 + α-Keto Acid 2 ⇌ α-Keto Acid 1 + Amino Acid 2

Example: Alanine aminotransferase (ALT) and Aspartate aminotransferase (AST) are clinically important transaminases.

• ALT Reaction: Alanine + α-Ketoglutarate ⇌ Pyruvate + Glutamate
• AST Reaction: Aspartate + α-Ketoglutarate ⇌ Oxaloacetate + Glutamate

Significance:

• Amino Acid Synthesis: Allows the synthesis of non-essential amino acids by transferring amino groups to appropriate α-keto acid precursors.
• Amino Acid Degradation: Collects amino groups from various amino acids into a common intermediate, glutamate, which can then be deaminated.
• Link to Central Metabolism: Connects amino acid metabolism to carbohydrate and lipid metabolism via pyruvate, oxaloacetate, and α-ketoglutarate, which are intermediates of glycolysis and the Krebs cycle.

Deamination

Deamination is the removal of an amino group from a molecule, often an amino acid, forming ammonia.

Definition: Deamination is the process by which an amino group is removed from an organic compound, most commonly an amino acid, resulting in the release of ammonia (NH₃) and the formation of a corresponding α-keto acid.

• Location: Primarily in the liver and kidneys.
• Main Type: Oxidative Deamination is the most common and significant type, particularly for glutamate.

Oxidative Deamination of Glutamate:

Glutamate is a central amino acid in amino group metabolism because it readily undergoes oxidative deamination, releasing ammonia. This reaction is catalyzed by glutamate dehydrogenase.

Glutamate + NAD(P)⁺ + H₂O → α-Ketoglutarate + NH₄⁺ + NAD(P)H

Important Note: The ammonia (NH₄⁺) produced is highly toxic and must be detoxified, primarily by conversion to urea in the urea cycle.

Significance:

• Ammonia Production: The primary source of ammonia for the urea cycle.
• Entry into Central Metabolism: The resulting α-keto acid (e.g., α-ketoglutarate from glutamate) can enter the Krebs cycle or be used for gluconeogenesis or fatty acid synthesis.

Urea Cycle

The Urea Cycle is the primary mechanism for the detoxification of ammonia in mammals, converting it into less toxic urea for excretion.

Definition: The Urea Cycle (also known as the Ornithine Cycle) is a metabolic pathway that converts excess ammonia (NH₃), a toxic byproduct of amino acid metabolism, into urea, a less toxic compound that can be safely excreted from the body via the kidneys.

• Location: Primarily in the liver. Two reactions occur in the mitochondrial matrix, and three in the cytosol.
• Purpose: To eliminate toxic ammonia, which can be detrimental to the central nervous system.

Key Substrates:

• Ammonia (NH₃, from deamination)
• Carbon Dioxide (CO₂, from cellular respiration)
• Aspartate (provides the second amino group)

Key Product:

• Urea (excreted)

Overview of Steps and Intermediates:

1. Formation of Carbamoyl Phosphate (Mitochondria):

   Ammonia (NH₄⁺) and CO₂ combine to form carbamoyl phosphate. This ATP-dependent reaction is catalyzed by Carbamoyl Phosphate Synthetase I and is the committed step.

2. Formation of Citrulline (Mitochondria):

   Carbamoyl phosphate reacts with Ornithine to form Citrulline. Ornithine is regenerated at the end of the cycle.

3. Formation of Argininosuccinate (Cytosol):

   Citrulline moves to the cytosol and reacts with Aspartate (which provides the second amino group) to form Argininosuccinate. This reaction consumes ATP.

4. Cleavage of Argininosuccinate (Cytosol):

   Argininosuccinate is cleaved to yield Arginine and Fumarate. Fumarate can be converted to malate and enter the Krebs cycle, linking the two cycles.

5. Hydrolysis of Arginine (Cytosol):

   Arginine is hydrolyzed by the enzyme Arginase to produce Urea and regenerate Ornithine. Ornithine is then transported back into the mitochondria to continue the cycle.

Urea Structure:

H₂N-CO-NH₂

Energy Cost:

The urea cycle is an energy-consuming process, requiring 3 ATP molecules (equivalent of 4 high-energy phosphate bonds) per molecule of urea synthesized.

Clinical Significance:

Defects in urea cycle enzymes can lead to hyperammonemia (high blood ammonia levels), which is very toxic, especially to the brain, and can cause severe neurological problems or death.