Biomolecules and Enzymes Notes

Recap

• Chemistry is used to influence biochemistry by leveraging intermolecular forces.

• Intermolecular forces like electrostatics, hydrogen bonds (HB), Van der Waals forces (VdW), and the hydrophobic effect are crucial in molecular interactions.

• Amino acids are the basic building blocks of proteins, each having unique properties.

• The 20 different amino acid side chains provide a wide range of intermolecular interactions, which are critical for protein structure and function.

• Acid/base equilibria (pKa) of amino acids determine their charge under biological conditions, which affects their interactions.

  • Lysine (Lys, K): pKa = 10.5 (conjugate acid); positively charged at physiological pH.

  • Glutamic acid (Glu, E): pKa = 4.1; negatively charged at physiological pH.

Peptides

• Amino acids are linked into sequence-defined polymers called peptides, with precise arrangement.

• The arrangement of side chains and their functionalities in space is determined by this sequence.

• Amino acid sequence dictates protein structure, which ultimately governs protein function and drug interactions; this relationship is central to biochemistry and pharmacology.

Writing Amino Acid Sequences

• Three amino acids in sequence form a tripeptide (e.g., Ser-Gly-Leu), following a standardized nomenclature.

• Sequences are always written from N-terminus (amine group) to C-terminus (carboxyl group), indicating directionality.

• Length classification:

  • < 50 residues = peptide, with oligopeptide being a smaller subset (< \sim10).

  • > 50 residues = polypeptide (typically unfolded), transitioning to protein upon folding.

Peptide Bonds

• Peptide bonds are amide bonds, formed through a specific chemical reaction.

• Amides result from a condensation reaction between amines and carboxylic acids, producing water as a byproduct.

• General amide formation: RCOOH + R'NH2 \rightarrow RCONHR' + H2O

• Amides can be primary, secondary, or tertiary, each with distinct properties and reactivity.

Amide Bond Structure

• The nitrogen lone pair is conjugated to the C=O double bond, resulting in a partial double-bond character between N and C, affecting its properties.

• The partial double bond restricts rotation about the C-N bond, making the group planar and influencing protein conformation.

Properties of Amides

• Amides are not basic, and amide nitrogens are not nucleophilic due to electron delocalization.

• Amines, in contrast, are basic, with nucleophilic amine nitrogens available for reactions.

• Amides exhibit stability under weakly acidic and basic conditions, distinguishing them from more labile esters.

• Hydrolysis of amides requires extreme conditions, highlighting their robustness in biological systems.

Amide Synthesis

• Amides are synthesized from activated analogues of carboxylic acids, enhancing reactivity for bond formation.

• Amines act as nucleophiles, attacking activated carboxylic acid derivatives acting as electrophiles.

  • Examples of activated carboxylic acids include acyl chloride and acid anhydride, which facilitate amide bond formation.

Amide Synthesis - DCC

• Dicyclohexylcarbodiimide (DCC) is a common activating agent enabling one-pot synthesis, simplifying procedures.

• Water is transferred onto DCC, yielding a urea side product, which must be removed from the reaction mixture.

Other Carbodiimides

• EDAC (N-Cyclohexyl-N′-(2-morpholinoethyl)carbodiimide methyl-p-toluenesulfonate) is water-soluble, facilitating its use in peptide synthesis, particularly after DCC activation.

• DIC (N,N′-Diisopropylcarbodiimide) serves as an alternative carbodiimide for activating carboxylic acids.

Peptide Synthesis Challenges

• Reacting unprotected amino acids results in a random polymer (polypeptide) lacking control over length or sequence, posing a significant challenge: A + B \rightarrow A-B-B-A-B-A-A-A-B-B-B-B-A-B-A-A-B-B

• Biology relies on peptides with precisely defined lengths and sequences for specific functions, necessitating controlled synthesis methods.

Protecting Groups (PGs)

• Protecting groups prevent unwanted reactions at undesired sites, ensuring selectivity in peptide synthesis.

• Purification at each step becomes increasingly laborious for long peptides (10s to 100s of amino acids), emphasizing the need for efficient synthetic strategies.

Automation of Peptide Synthesis

• Solid-phase peptide synthesis enables automation, enhancing efficiency and reproducibility.

• Key steps include:

  1. Coupling reagent activation.

  2. Cleavage from the solid support (bead).

  3. Repetitive coupling reagent addition.

  4. Deprotection of the amine protecting group (PG).

  5. Washing away excess reactants and byproducts.

Writing Sequences

• Examples of peptide sequences:

  • Tyr-Gly-Gly-Phe-Leu (YGGFL).

  • AcTyr-Gly-Gly-Phe-LeuNH2 (AcYGGFLNH2), where modifications like acetylation (Ac) and amidation (NH2) are specified.

• Nomenclature:

  • Dipeptide = 2 amino acids; Tripeptide = 3 amino acids; Tetrapeptide = 4 amino acids, etc.

Isoelectric Point (pI) Calculation

• The isoelectric point (pI) is the pH at which a molecule carries no net electrical charge, crucial for understanding protein behavior.

1. List pKa values from highest to lowest for the peptide's ionizable groups.

2. Choose a pH below the lowest pKa and calculate the net charge.

3. Select a pH between the first and second lowest pKa values, and recalculate the net charge.

4. Repeat this process for all pH intervals.

5. The pI is the average of the pKa values flanking the point where the net charge (q) equals zero.

• Example:

  • pKa values: 9.2, 3.7, 2.4

  • pI = (3.7 + 2.4) / 2 = 3.1

Biological Activity of Peptides

• Primary structure refers to the residue sequence alone, dictating potential interactions and folding.

• Protein is a folded polypeptide chain exhibiting complex three-dimensional structure and function.

Signalling Peptides

• Peptides are essential in biological signaling pathways, mediating a range of physiological effects.

  • Gastrin: LEEEEEAYGWMDF-NH2 (Stimulates HCl secretion in the stomach).

  • Oxytocin: CYIQNCPLG-NH2 (Induces uterine contractions).

  • Vasopressin: CYFQNCPRG-NH2 (Causes vasoconstriction, acts as an antidiuretic).

  • Bradykinin: RPPGFSPFR (Promotes vasodilation).

• Opioid Receptors:

  • β-Endorphin: YGGFMTSEKSQTPLVTLFKNAIIKNAYKKGE.

  • Enkephalin: YGGFM.

  • β-Casomorphin 1-3: YPF.

  • Gluten exorphin B5: YGGWL.

• Other Signalling Peptides:

  • Aspartame = Asp-Phe-OMe (Artificial sweetener).

  • RGD (Promotes cell surface adhesion).

  • KDEL (Targets proteins to the endoplasmic reticulum).

  • KVLKKRR (Targets proteins to the nucleus).

  • PPKKKRKV (Targets proteins to the nucleus).

  • KMSVLTPLLLRGLTGSARRLPVPRAKC (Targets proteins to the mitochondria).

Forensic Proteomics

• Glendon J. Parker, Heather E. McKiernan, Kevin M. Legg, Zachary C. Goecker, Forensic proteomics, Forensic Science International: Genetics, Volume 54, 2021, 102529.

Peptides - Summary + Context

• Peptides are chains of amino acids arranged in a specific sequence, determining their properties.

• Peptide sequences arrange potential intermolecular interactions (from side chains) in space, influencing their biological activity.

• Amino acids are linked by amide bonds, which possess a fixed planar geometry and high stability.

• Amide bonds form through the reaction of an amine with an activated carboxylic acid derivative.

• Peptides are biologically significant as building blocks of proteins, which are long, folded polypeptides.

• Short peptides often participate directly in biological signaling pathways, acting as hormones or neurotransmitters.