Biochemistry Study Notes: Enzyme Mechanisms, Lipids, Membranes, Carbohydrates, and Metabolism

Chymotrypsin & Enzyme Inhibition

Chymotrypsin Overview

Chymotrypsin is a digestive enzyme (serine protease) secreted by the pancreas, crucial for protein digestion in the small intestine.

• Type: Digestive enzyme (serine protease)

• Molecular Weight: ~25–30 kDa

• Cleaves: Peptide bonds on the C-terminal side of aromatic residues: Phenylalanine (F), Tryptophan (W), Tyrosine (Y)

• Catalytic Triad:

  • Asp102: Stabilizes charge on His57

  • His57: Acts as acid/base

  • Ser195: Nucleophile that attacks peptide bond

• Active Site Pocket: Hydrophobic—fits aromatic residues

Catalytic Mechanism (Two-Phase Reaction)

The catalytic mechanism of chymotrypsin involves two main phases: acylation and deacylation, each with distinct steps.

Phase 1: Acylation

1. Substrate Binding:

   • Substrate fits into hydrophobic pocket.

   • Asp102–His57 interaction stabilizes histidine’s charge.

   • Ser195 hydrogen bonds with His57 and substrate’s carbonyl.

2. Nucleophilic Attack:

   • His57 deprotonates Ser195 → forms alkoxide ion (O–).

   • Alkoxide attacks substrate C=O → forms tetrahedral intermediate.

3. Collapse & Cleavage:

   • Intermediate collapses → peptide bond breaks.

   • Acyl-enzyme intermediate remains (Ser195 covalently linked to substrate).

Phase 2: Deacylation

1. Water Activation:

   • H2O enters; His57 deprotonates it → forms OH–.

2. Second Nucleophilic Attack:

   • OH– attacks the acyl-enzyme C=O → forms second tetrahedral intermediate.

3. Product Release:

   • Intermediate collapses → breaks Ser195–substrate bond.

   • Product released; enzyme restored to native state.

Irreversible Inhibition Example

• DIFP (Diisopropyl fluorophosphate): Covalently binds to Ser195, permanently inactivating the enzyme.

Enzyme Inhibition Types

Enzyme inhibitors reduce or abolish enzyme activity by various mechanisms. The main types are compared below:

Inhibition Type	Binds To	Active Site?	Effect on Vmax	Effect on Km	Lineweaver-Burk Plot Feature
Competitive	E only	Yes	Unchanged	↑ Increases	Lines intersect y-axis
Uncompetitive	ES only	No	↓ Decreases	↓ Decreases	Parallel lines
Mixed	E or ES	Yes/No	↓ or –	↑ or ↓	Intersect left of y-axis
Irreversible	E (covalently)	Yes	Eliminates activity	–	–

Kinetic Equations

• Competitive: $V_0 = \frac{V_{max}[S]}{\alpha K_m + [S]}, \quad \alpha = 1 + \frac{[I]}{K_I}$

• Uncompetitive: $V_0 = \frac{V_{max}[S]}{K_m + \alpha'[S]}, \quad \alpha' = 1 + \frac{[I]}{K_{I'}}$

• Mixed: $V_0 = \frac{V_{max}[S]}{\alpha K_m + \alpha'[S]}$ $\alpha = 1 + \frac{[I]}{K_I}, \quad \alpha' = 1 + \frac{[I]}{K_{I'}}$

Lipids

Galactolipids

Galactolipids are major components of plant membranes, especially in chloroplasts.

• Structure: 1–2 galactose residues linked to C-3 of 1,2-diacylglycerol.

• Location: Plant chloroplast membranes.

• Function: Environmental adaptation in plants.

Sphingolipids

• Structure: Polar head & 2 nonpolar tails.

• Backbone: Sphingosine (no glycerol).

• Function: Membrane stability, neural tissue structure.

• Fatty acid linked to sphingosine at C-2 via amide linkage.

• Analogous to diacylglycerol.

Cell Recognition

• Glycosphingolipids = cell surface recognition sites.

• Blood Groups (A, O, B): Determined by glycosphingolipid head groups.

Sterols

• Structure: 4 fused hydrocarbon rings (steroid nucleus).

• Function: Maintain membrane fluidity and structure.

Cholesterol (Animals)

• Amphipathic: Polar OH head + hydrophobic tail.

• Analogs:

  • Plants — Stigmasterol

  • Fungi — Ergosterol

Sterol Derivatives

• Steroid hormones: Regulate gene expression.

• Bile acids: Derived from cholesterol; emulsify dietary fats.

Lipid Breakdown

• Occurs in lysosomes.

• Phospholipases A — remove one fatty acid.

• Lysophospholipases — remove remaining FA.

• Glycosidases — remove sugars from gangliosides.

Lipases

• Hydrolyze stored triacylglycerols → release fatty acids for energy.

• Found in adipocytes and germinating seeds.

Membrane Structure & Function

Membrane Protein Roles

• Transporters: Move solutes across membranes.

• Receptors: Receive and transmit signals.

• Ion Channels: Conduct electrical impulses.

• Adhesion Molecules: Connect neighboring cells.

Membrane Dynamics

Membranes are fluid, allowing lipid and protein movement. Transbilayer movement (flip-flop) is slow and catalyzed by enzymes.

Enzyme	Direction	Energy Use	Notes
Flippase	Outer → Inner (PE, PS)	ATP-dependent	Maintains leaflet asymmetry
Floppase	Inner → Outer (PC, cholesterol, sterols)	ATP-dependent	–
Scramblase	Bidirectional	No ATP	Randomizes lipid distribution (Ca2+-activated)

Transporter Proteins

• Reduce ΔG by creating hydrophilic pathways.

• Passive Transport: Down concentration gradient.

• Active Transport: Against gradient; requires energy.

Ion Channels

• Provide aqueous pores for ions.

• Gated: Open/close in response to signals.

• Ion-Selective: Flow stops when gradient or gate closes.

GLUT1 (Glucose Transporter)

• Location: Erythrocytes (RBCs).

• Structure: 12 α-helical segments (amphipathic).

• Conformations:

  • T1: Faces outside

  • T2: Faces inside

• Kinetic Equation: $V_0 = \frac{V_{max}[S]_{out}}{K_t + [S]_{out}}$

Carbohydrates

Aldoses vs. Ketoses

• Aldose: Carbonyl at chain end → aldehyde.

• Ketose: Carbonyl internal → ketone.

Hemiacetals & Hemiketals

• Formed by reaction of an alcohol + aldehyde/ketone.

• First step in ring formation of sugars.

Pyranoses & Furanoses

• Pyranose: 6-membered ring (C-1 → C-5 in glucose).

• Furanose: 5-membered ring (C-2 → C-5 in fructose).

α and β Anomers

• Anomeric carbon: Carbon derived from the carbonyl.

• α: OH on anomeric carbon opposite CH2OH.

• β: OH on same side.

• In solution: Glucose ≈ 36% α, 64% β.

Glycosidic Bonds

• O-glycosidic bond: Between anomeric carbon and hydroxyl group of another sugar.

• Acid-labile; defines sugar linkages.

Polysaccharides

Type	Linkages	Example	Notes
Homopolysaccharide	One sugar type	Starch, Glycogen	Energy storage
Heteropolysaccharide	Multiple sugars	Peptidoglycan	Structure

• Starch (Plants):

  • Amylose: Linear (α1→4)

  • Amylopectin: Branched (α1→4, α1→6)

• Glycogen (Animals/Fungi):

  • Highly branched (every 8–12 residues)

  • More compact and soluble than starch

Metabolism Overview

Catabolism

• Degradative; releases energy (exergonic).

• Pathways converge to central intermediates (e.g., Acetyl-CoA).

Anabolism

• Biosynthetic; requires energy (endergonic).

• Pathways diverge to build macromolecules.

ATP: The Energy Currency

• ATP hydrolysis provides energy for anabolic reactions.

Mg2+ and ATP

• Mg2+ binds ATP/ADP to neutralize charge.

• True substrate in most reactions = MgATP2–.