Enzyme Inhibition

Overview

Enzyme inhibition refers to the process by which the activity of an enzyme is decreased or stopped by a specific molecule called an inhibitor. Understanding the types and mechanisms of inhibition is crucial for biochemistry, especially in drug design and metabolic regulation.

Competitive Inhibition

• Mechanism: Inhibitor competes with substrate for the active site of the enzyme.

• Effect on Kinetics:

  • Vmax: Unchanged

  • Km: Increases (more substrate needed to reach half-maximal velocity)

• Lineweaver–Burk (L-B) Plot:

  • Lines intersect at the y-axis (same 1/Vmax)

  • Different slopes (αKm/Vmax)

• Equation: where

Uncompetitive Inhibition

• Mechanism: Inhibitor binds only to the enzyme–substrate (ES) complex, not to free enzyme.

• Binding Site: Distinct from active site.

• Effect on Kinetics:

  • Vmax: Decreases

  • Km: Decreases

  • Km/Vmax: Constant

• Lineweaver–Burk Plot:

  • Parallel lines (same slope)

• Equation: where

Mixed Inhibition

• Mechanism: Inhibitor binds to both free enzyme (E) and the ES complex at a site distinct from the active site.

• Effect on Kinetics:

  • Vmax: Decreases

  • Km: Can increase or decrease, depending on inhibitor preference

• Lineweaver–Burk Plot:

  • Lines intersect left of y-axis (not parallel)

  • Different slopes and y-intercepts

• Equation: where ,

Irreversible Inhibition

• Mechanism: Inhibitor covalently binds to or destroys a key enzyme functional group, permanently inactivating it.

• Example: DIFP (Diisopropyl fluorophosphate) binds covalently to serine proteases, inactivating the Ser195 residue in enzymes like chymotrypsin.

Summary Table – Inhibition Types

Chymotrypsin Mechanism

Overview

Chymotrypsin is a digestive enzyme (serine protease) from the pancreas. It cleaves peptide bonds on the C-terminal side of aromatic residues (Trp, Phe, Tyr). The catalytic triad consists of Asp102, His57, and Ser195, with His57 acting as both acid and base during catalysis.

Catalytic Mechanism (Two Phases)

Phase 1 – Acylation (Formation of Acyl-Enzyme Intermediate)

1. Substrate Binding: Substrate binds in hydrophobic pocket; His57–Asp102 interaction stabilizes the charge.

2. Nucleophilic Attack: Ser195 donates H⁺ to His57, forming a nucleophilic alkoxide ion (–O⁻). This attacks the substrate C=O, breaking its bond and forming a tetrahedral intermediate.

3. Peptide Bond Cleavage: Tetrahedral intermediate collapses, C–O⁻ reforms the C=O bond, peptide bond breaks, forming the acyl-enzyme intermediate. The N-terminal fragment of the substrate gains a proton from His57 and leaves.

Phase 2 – Deacylation (Release of Product, Regeneration of Enzyme)

1. Water Activation: Water enters active site; His57 deprotonates H₂O, forming OH⁻ (strong nucleophile), which attacks the C=O of the acyl-enzyme intermediate.

2. Second Tetrahedral Intermediate: Attack forms unstable tetrahedral intermediate; C=O bond breaks, forming C–O⁻.

3. Collapse and Product Release: Intermediate collapses, C–O⁻ reforms the C=O bond, releasing the product (peptide with COOH terminus). Enzyme is regenerated (Ser195 restored).

Lipids

Lipids Overview

Lipids are a diverse group of hydrophobic biomolecules essential for membrane structure, energy storage, and signaling. Major classes include galactolipids, phospholipids, sphingolipids, sterols, and lipoproteins.

Galactolipids

• Structure: Contain one or two galactose residues linked by a glycosidic bond to C-3 of a 1,2-diacylglycerol.

• Location: Predominantly in plant cells.

• Function/Adaptation: Along with ether-linked lipids of archaea, galactolipids help organisms adapt to environmental conditions.

Phospholipids and Glycolipids

• Structure: Large class of membrane phospholipids and glycolipids; contain a polar head group and two nonpolar tails; no glycerol backbone; built on sphingosine (a long-chain amino alcohol) or its derivatives.

• Function: Important components of cell membranes, especially in the nervous system.

Ceramides

• Definition: The structural parent molecule of all sphingolipids.

• Structure: C-1, C-2, C-3 of sphingosine are analogous to the three carbons of glycerol in glycerophospholipids. A fatty acid is attached to the –NH on C-2 via an amide linkage.

• Comparison: Structurally similar to diacylglycerol.

Sphingolipids and Cell Recognition

• Role: Serve as sites of biological recognition on cell surfaces.

• Clinical Significance: Human blood groups (O, A, B) are determined by the oligosaccharide head groups of specific glycosphingolipids.

Sterols

• Function: Structural lipids present in membranes of most eukaryotic cells.

• Structure: Contain a steroid nucleus (four fused rings); almost planar and rigid.

Cholesterol

• Major sterol in animal tissues.

• Amphipathic: Polar head group + nonpolar hydrocarbon body.

• Function: Essential membrane constituent.

• Analogs: Stigmasterol in plants, ergosterol in fungi.

Sterol Derivatives with Biological Activity

• Steroid hormones: Regulate gene expression.

• Polar cholesterol derivatives: Emulsify dietary fats in the intestine, making them accessible to digestive lipases.

Lipases

• Function: Enzymes that hydrolyze stored triacylglycerols, releasing fatty acids for energy use.

• Location: Found in adipocytes (fat cells) and germinating seeds.

Lipid Degradation in Lysosomes

• Phospholipids and sphingolipids are broken down in lysosomes.

• Enzymes involved:

  • Phospholipases A: Remove one fatty acid.

  • Lysophospholipases: Remove the remaining fatty acid.

  • Lysosomal enzymes: Remove sugar units stepwise from gangliosides.

Membranes

Functions of Membrane Proteins

• Transporters: Move specific organic solutes and inorganic ions across the membrane.

• Receptors: Detect extracellular signals and trigger molecular changes inside the cell.

• Ion Channels: Mediate electrical signaling between cells.

Membrane Dynamics

• Membranes are fluid.

• Transbilayer movement of lipids (flip-flop) is slow and requires catalysis.

Flippases

• Catalyze translocation of phosphatidylethanolamine (PE) and phosphatidylserine (PS) from extracellular to cytoplasmic leaflet.

• ATP-dependent (~1 ATP per phospholipid).

Floppases

• Move phospholipids and sterols from cytoplasmic to extracellular leaflet.

• ATP-dependent.

Scramblases

• Move any membrane phospholipid across the bilayer down its concentration gradient.

• Not ATP-dependent; some require Ca2+.

• Cause controlled randomization of lipid head-group composition between leaflets.

Transporter Proteins (Transmembrane Transport)

• Reduce activation energy (ΔGt) for diffusion by:

  • Forming noncovalent interactions with the dehydrated solute.

  • Providing a hydrophilic transmembrane pathway.

• Active transporters: Move solutes against their concentration or electrical gradient (require energy).

Ion Channels

• Provide an aqueous path for inorganic ions to diffuse rapidly.

• Usually gated (regulated by biological signals).

• Ion-selective.

• Flow stops when gate closes or electrochemical gradient disappears.

GLUT1 (Glucose Transporter)

• Integral membrane protein with 12 hydrophobic α-helices (amphipathic).

• Transports glucose in erythrocytes (RBCs).

• Two conformations:

  • T1: Glucose-binding site faces outside.

  • T2: Glucose-binding site faces inside.

Enzyme-like Function of GLUT1

• Functions analogously to an enzyme-catalyzed reaction:

• Substrate (Sout): Glucose outside cell.

• Product (Sin): Glucose inside cell.

• Transporter (T): Alternates between T1 and T2 forms.

• Kinetics equation: where is the initial velocity of glucose uptake, is the transport constant.