Peptide Bonds and Biochemical Structure

Written by Dr. Johnathon Anderson, Ph.D., a research scientist specializing in regenerative medicine and serving as an Associate Professor at the University of California Davis School of Medicine

Peptide Bonds and Biochemical Structure

Amino acids serve as the foundational building blocks of peptides and proteins. While we’ve previously explored their structural nuances, classifications, and protonation behaviors, this discussion will focus on how amino acids join via peptide bonds, how these bonds can be broken, and the unique resonance properties that give peptide bonds their distinct strength.

Peptide Bond Formation and Hydrolysis

Formation: Peptide bond creation is a condensation reaction, where two amino acids are joined, and a water molecule is released as a byproduct. The lone pair on the nitrogen atom of an amine group acts as a nucleophile, attacking the carbonyl carbon of a neighboring amino acid's carboxyl group. However, this reaction doesn’t happen spontaneously due to two major barriers:

1. Thermodynamic Barrier

   Peptide bond formation is endergonic, requiring an input of energy. In biological systems, this challenge is overcome by coupling the reaction to exergonic processes, such as ATP hydrolysis.

2. Kinetic Barrier

   The reaction’s high activation energy is reduced by biological catalysts—enzymes that facilitate the process efficiently.

Hydrolysis: Breaking a peptide bond is essentially the reverse of its formation, involving a hydrolysis reaction where water is consumed. This reaction is exergonic, releasing energy, but it occurs very slowly in biological systems without enzymatic help. Proteases are specialized enzymes that catalyze peptide bond hydrolysis, accelerating the breakdown of proteins.

Resonance and the Strength of Peptide Bonds

Peptide bonds possess unique stability due to their resonance hybrid structure. While typically depicted as a single bond between the carbonyl carbon and the amine nitrogen, the true nature of a peptide bond lies between two forms (Figure 4):

1. A double-bonded carbonyl group with a single bond to the amine nitrogen.

2. A resonance structure where the lone pair on the nitrogen forms a double bond with the carbon, pushing the oxygen to carry a negative charge.

Although the left-hand structure dominates, this resonance gives the peptide bond a partial double-bond character. This results in:

• Rigidity: Restricted rotation around the bond.

• Strength: Greater stability compared to a typical single bond.

Order Matters: N-terminus and C-terminus

In peptides, the sequence of amino acids determines their structure and function. Each peptide has two ends:

• N-terminus: The free amine group of the first amino acid.

• C-terminus: The free carboxyl group of the last amino acid.

The order of these termini is critical. For example, a dipeptide with alanine at the N-terminus and tyrosine at the C-terminus (N-Ala-Tyr-C) differs structurally and functionally from one where tyrosine is at the N-terminus and alanine at the C-terminus (N-Tyr-Ala-C).

Peptides are fundamental to numerous biological and biochemical processes, acting as key components in cellular signaling, immune defense, and structural frameworks. Chemically, peptides are defined as short chains of amino acids (2–50 residues) linked by covalent peptide bonds formed through condensation reactions. These bonds result from a reaction between the carboxyl group of one amino acid and the amino group of another, releasing a molecule of water in the process.

Structural Classification of Peptides

Peptides are categorized based on the number of amino acid residues in their chain:

• Oligopeptides: Chains containing 10–20 amino acids.

• Polypeptides: Chains exceeding 20 amino acids, often serving as precursors to fully folded proteins.

Each amino acid residue is the molecular remnant of the original amino acid after dehydration. The sequence and structure of amino acid residues within a peptide dictate its functional properties. Peptide chains are synthesized in vivo via a stepwise process in which new residues are added to the amino-terminal end, elongating the chain in a sequential manner.

Peptide Bond Characteristics

The peptide bond itself is notable for its partial double-bond character, attributed to resonance between the carbonyl oxygen and the amide nitrogen. This gives the bond increased rigidity, restricting rotation around the C–N axis and enforcing a planar configuration. Steric hindrance from side chains (R-groups) further influences the bond’s geometry, favoring the trans isomer. The surrounding bonds in the peptide backbone, however, retain rotational freedom, enabling the formation of secondary and tertiary structures essential for biological activity.

Bioactive Peptides: Applications in Therapeutics and Biochemistry

Peptides serve not only as building blocks of proteins but also as active agents with diverse biochemical roles. Bioactive peptides are specifically designed or derived sequences with therapeutic potential, influencing physiological pathways including:

• Antimicrobial activity: Disrupting pathogenic membranes.

• Anti-inflammatory effects: Modulating immune responses.

• Antioxidant properties: Reducing oxidative stress.

• Hormonal regulation: Acting as endocrine or paracrine signaling molecules.

The pharmaceutical industry leverages these properties to design peptide-based biologics, mimicking endogenous molecules for targeted therapeutic effects.

Cellular Synthesis and Processing of Peptides

Ribosomal Peptide Synthesis

Peptide biosynthesis occurs through translation, a cellular process in which messenger RNA (mRNA) templates guide the ribosomal assembly of amino acid chains. The steps include:

1. Initiation: The small ribosomal subunit binds to the mRNA start codon, AUG, aligning it with the anticodon of initiator tRNA carrying methionine.

2. Elongation: Successive amino acids are added to the growing chain via peptide bonds catalyzed by the ribosome’s peptidyl transferase center.

3. Termination: Upon encountering a stop codon, the nascent peptide is released for further processing.

Post-translational modifications such as phosphorylation, glycosylation, and acetylation refine peptide function and localization. For instance, preprohormones are cleaved into prohormones and subsequently into active peptides within the endoplasmic reticulum and Golgi apparatus.

Laboratory Synthesis: Solid-Phase Peptide Synthesis (SPPS)

In vitro, synthetic peptides are produced using SPPS. This method allows precise assembly of peptide chains through iterative cycles of amino acid coupling and deprotection, achieving sequences with high specificity and yield.

Molecular Functions of Peptide Hormones

Peptide hormones are pivotal in maintaining homeostasis and coordinating physiological responses. These molecules, ranging from small oligopeptides to complex polypeptides, interact with receptors to trigger intracellular signaling cascades. Key examples include:

• Insulin: Regulates glucose uptake and metabolism.

• Glucagon: Counteracts hypoglycemia by mobilizing stored glucose.

• Calcitonin Gene-Related Peptide (CGRP): Modulates vasodilation and energy homeostasis.

Synthetic analogs of these hormones are critical in managing conditions such as diabetes and cardiovascular disorders.

Pathophysiological Implications and Clinical Applications

Peptide-Receptor Complexes

Peptides often act through specific receptors, such as G-protein-coupled receptors (GPCRs), to regulate cellular functions. Dysregulation of these interactions can result in pathological states, including oncogenesis and metabolic disorders. Advances in peptide engineering have enabled the creation of targeted therapies that disrupt aberrant peptide-receptor interactions or exploit them for therapeutic benefit.

Antimicrobial Peptides (AMPs)

As components of innate immunity, AMPs like defensins and cathelicidins are critical in pathogen defense. These peptides interact with microbial membranes, often disrupting their integrity. Research into AMPs continues to uncover their potential as novel antibiotics in an era of rising antimicrobial resistance.

References

1. FRIEDBERG F, WINNICK T, GREENBERG DM. Peptide synthesis in vivo. J Biol Chem. 1947 Aug;169(3):763. [PubMed]

2. Davidovich C, Belousoff M, Bashan A, Yonath A. The evolving ribosome: from non-coded peptide bond formation to sophisticated translation machinery. Res Microbiol. 2009 Sep;160(7):487-92. [PubMed]

3. Lovejoy DA, Hogg DW, Dodsworth TL, Jurado FR, Read CC, D'Aquila AL, Barsyte-Lovejoy D. Synthetic Peptides as Therapeutic Agents: Lessons Learned From Evolutionary Ancient Peptides and Their Transit Across Blood-Brain Barriers. Front Endocrinol (Lausanne). 2019;10:730. [PMC free article] [PubMed]

4. Saladin PM, Zhang BD, Reichert JM. Current trends in the clinical development of peptide therapeutics. IDrugs. 2009 Dec;12(12):779-84. [PubMed]

5. Hanson G, Alhusaini N, Morris N, Sweet T, Coller J. Translation elongation and mRNA stability are coupled through the ribosomal A-site. RNA. 2018 Oct;24(10):1377-1389. [PMC free article] [PubMed]

6. Kleinkauf H, von Döhren H. Applications of peptide synthetases in the synthesis of peptide analogues. Acta Biochim Pol. 1997;44(4):839-47. [PubMed]

7. Albericio F, Lloyd-Williams P, Giralt E. Convergent solid-phase peptide synthesis. Methods Enzymol. 1997;289:313-36. [PubMed]

8. Barany G, Merrifield RB. A chromatographic method for the quantitative analysis of the deprotection of dithiasuccinoyl (Dts) amino acids. Anal Biochem. 1979 May;95(1):160-70. [PubMed]

9. Sanders EJ, Harvey S. Peptide hormones as developmental growth and differentiation factors. Dev Dyn. 2008 Jun;237(6):1537-52. [PubMed]

10. Harris RM, Dijkstra PD, Hofmann HA. Complex structural and regulatory evolution of the pro-opiomelanocortin gene family. Gen Comp Endocrinol. 2014 Jan 01;195:107-15. [PubMed]

11. Chan WY. An investigation of the natriuretic, antidiuretic and oxytocic actions of neurohypophysial hormones and related peptides: delineation of separate mechanisms of action and assessment of molecular requirements. J Pharmacol Exp Ther. 1976 Mar;196(3):746-57. [PubMed]

12. McLaughlin MB, Jialal I. StatPearls [Internet]. StatPearls Publishing; Treasure Island (FL): Jul 17, 2023. Biochemistry, Hormones. [PubMed]

13. Sandoval DA, D'Alessio DA. Physiology of proglucagon peptides: role of glucagon and GLP-1 in health and disease. Physiol Rev. 2015 Apr;95(2):513-48. [PubMed]

14. Chey WY, Chang TM. Secretin: historical perspective and current status. Pancreas. 2014 Mar;43(2):162-82. [PubMed]

15. Brandler J, Miller LJ, Wang XJ, Burton D, Busciglio I, Arndt K, Harmsen WS, Camilleri M. Secretin effects on gastric functions, hormones and symptoms in functional dyspepsia and health: randomized crossover trial. Am J Physiol Gastrointest Liver Physiol. 2020 Apr 01;318(4):G635-G645. [PMC free article] [PubMed]

16. Poyner DR. Calcitonin gene-related peptide: multiple actions, multiple receptors. Pharmacol Ther. 1992;56(1):23-51. [PubMed]

17. Sanford D, Luong L, Gabalski A, Oh S, Vu JP, Pisegna JR, Germano P. An Intraperitoneal Treatment with Calcitonin Gene-Related Peptide (CGRP) Regulates Appetite, Energy Intake/Expenditure, and Metabolism. J Mol Neurosci. 2019 Jan;67(1):28-37. [PMC free article] [PubMed]

18. Dubowchik GM, Conway CM, Xin AW. Blocking the CGRP Pathway for Acute and Preventive Treatment of Migraine: The Evolution of Success. J Med Chem. 2020 Jul 09;63(13):6600-6623. [PubMed]

19. Deen M, Correnti E, Kamm K, Kelderman T, Papetti L, Rubio-Beltrán E, Vigneri S, Edvinsson L, Maassen Van Den Brink A., European Headache Federation School of Advanced Studies (EHF-SAS). Blocking CGRP in migraine patients - a review of pros and cons. J Headache Pain. 2017 Sep 25;18(1):96. [PMC free article] [PubMed]

20. Chopra S, Cherian D, Verghese PP, Jacob JJ. Physiology and clinical significance of natriuretic hormones. Indian J Endocrinol Metab. 2013 Jan;17(1):83-90. [PMC free article] [PubMed]

21. Rubattu S, Volpe M. Natriuretic Peptides in the Cardiovascular System: Multifaceted Roles in Physiology, Pathology and Therapeutics. Int J Mol Sci. 2019 Aug 16;20(16) [PMC free article] [PubMed]

22. Foster SR, Hauser AS, Vedel L, Strachan RT, Huang XP, Gavin AC, Shah SD, Nayak AP, Haugaard-Kedström LM, Penn RB, Roth BL, Bräuner-Osborne H, Gloriam DE. Discovery of Human Signaling Systems: Pairing Peptides to G Protein-Coupled Receptors. Cell. 2019 Oct 31;179(4):895-908.e21. [PMC free article] [PubMed]

23. Radaic A, de Jesus MB, Kapila YL. Bacterial anti-microbial peptides and nano-sized drug delivery systems: The state of the art toward improved bacteriocins. J Control Release. 2020 May 10;321:100-118. [PubMed]

24. Cole JN, Nizet V. Bacterial Evasion of Host Antimicrobial Peptide Defenses. Microbiol Spectr. 2016 Feb;4(1) [PMC free article] [PubMed]

25. Schittek B, Hipfel R, Sauer B, Bauer J, Kalbacher H, Stevanovic S, Schirle M, Schroeder K, Blin N, Meier F, Rassner G, Garbe C. Dermcidin: a novel human antibiotic peptide secreted by sweat glands. Nat Immunol. 2001 Dec;2(12):1133-7. [PubMed]

26. Gallo RL, Ono M, Povsic T, Page C, Eriksson E, Klagsbrun M, Bernfield M. Syndecans, cell surface heparan sulfate proteoglycans, are induced by a proline-rich antimicrobial peptide from wounds. Proc Natl Acad Sci U S A. 1994 Nov 08;91(23):11035-9. [PMC free article] [PubMed]

27. Park K, Lee S, Lee YM. Sphingolipids and antimicrobial peptides: function and roles in atopic dermatitis. Biomol Ther (Seoul). 2013 Jul 30;21(4):251-7. [PMC free article] [PubMed]

28. Marcinkiewicz M, Majewski S. The role of antimicrobial peptides in chronic inflammatory skin diseases. Postepy Dermatol Alergol. 2016 Feb;33(1):6-12. [PMC free article] [PubMed]

29. Lee S, Xie J, Chen X. Peptide-based probes for targeted molecular imaging. Biochemistry. 2010 Feb 23;49(7):1364-76. [PMC free article] [PubMed]