Peptidases: The Enzymes That Break Peptide Bonds

Every peptide bond can, in principle, be broken by water, but on its own that hydrolysis is extremely slow at neutral pH. Cells and laboratories alike depend on enzymes that accelerate it enormously and with considerable selectivity. A peptidase is an enzyme that catalyses the hydrolysis of peptide bonds, whether it trims single residues from the end of a chain or cuts a protein in the middle. Peptidases process hormone precursors, recycle proteins, activate clotting and digestive cascades, and terminate peptide signals.

For anyone working with synthetic peptides, peptidases are also a practical concern. Serum-containing culture media, tissue extracts and cell surfaces all carry peptidase activity that can degrade a test compound during an experiment. This article covers how peptidases are named and classified, their catalytic mechanisms, the DPP-4 cleavage of GLP-1 as a worked example, and how to control peptidase activity in assays.

Peptidase vs Protease: Terminology

The terms overlap. Protease is the older, more familiar word and is often used for enzymes that cut proteins internally, while peptidase is the umbrella term recommended by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology for all enzymes that hydrolyse peptide bonds, grouped under EC 3.4. In practice the two are used almost interchangeably. The MEROPS database, maintained since the 1990s, classifies peptidases by catalytic type and then by evolutionary family and clan, which has become the standard reference framework. Chemically, every sequence in our peptide catalog is a potential peptidase substrate; differences in sequence and modification determine how quickly a given enzyme can act on it.

Exopeptidases and Endopeptidases

The first practical distinction is where an enzyme cuts. Exopeptidases remove residues from a chain terminus and require a free terminal group, while endopeptidases cleave internal bonds. Cleavage positions are described with Schechter–Berger nomenclature: residues on the N-terminal side of the scissile bond are P1, P2 and so on, and those on the C-terminal side are P1′, P2′.

Class	Cleaves	Examples
Aminopeptidase	Single N-terminal residue	Aminopeptidase N, leucine aminopeptidase
Dipeptidyl peptidase	N-terminal dipeptide	DPP-4
Carboxypeptidase	Single C-terminal residue	Carboxypeptidases A and B
Peptidyl-dipeptidase	C-terminal dipeptide	Angiotensin-converting enzyme
Endopeptidase	Internal bonds	Trypsin, chymotrypsin, pepsin, thermolysin

Endopeptidase specificity is usually defined at P1. Trypsin cuts after lysine or arginine, typically not when proline follows; chymotrypsin prefers bulky aromatic residues; pepsin, active at acidic pH, favours hydrophobic residues on both sides of the bond. Terminal capping, by N-acetylation or C-amidation, removes the free groups that exopeptidases recognise. Whether such caps and other modifications are actually present in a synthetic lot is confirmed by mass spectrometry, alongside HPLC purity analysis, as outlined on our quality testing page.

Catalytic Mechanisms

Serine and threonine peptidases

Serine peptidases such as trypsin and chymotrypsin use a catalytic triad of serine, histidine and aspartate. The histidine activates the serine hydroxyl, which attacks the carbonyl carbon of the scissile bond; an oxyanion hole stabilises the tetrahedral intermediate, an acyl-enzyme forms, and water then releases the product. The proteasome uses a related strategy with an N-terminal threonine as the nucleophile.

Cysteine peptidases

Cysteine peptidases, including papain, the lysosomal cathepsins B and L and the caspases, use a cysteine thiol activated by histidine and pass through a thioester intermediate. Caspases are notably specific, cleaving after aspartate residues.

Aspartic and metallopeptidases

Aspartic peptidases such as pepsin, renin and cathepsin D use two aspartate residues to activate a water molecule directly, without a covalent enzyme intermediate. Metallopeptidases do the same with a bound metal ion, usually zinc, often coordinated through an HExxH motif; thermolysin, carboxypeptidase A, angiotensin-converting enzyme and neprilysin belong here. A small glutamic peptidase family also exists in fungi.

DPP-4 and GLP-1: A Research Example

Dipeptidyl peptidase-4 (DPP-4, also known as CD26) is a serine exopeptidase present on many cell surfaces and in a soluble circulating form. It removes N-terminal dipeptides when the second residue is proline or alanine. Native glucagon-like peptide-1, GLP-1(7–36) amide, begins His-Ala, so DPP-4 cleaves it to GLP-1(9–36) amide, a fragment with much lower activity at the GLP-1 receptor; the related hormone GIP is processed the same way. As a result, native GLP-1 persists in vivo for only minutes, as discussed in our overview of what is GLP-1.

Synthetic analogues show how sequence design addresses this. Semaglutide replaces alanine at position 8 with α-aminoisobutyric acid, a residue DPP-4 does not accept, and tirzepatide likewise carries Aib at its second position. Confirming that such substitutions are present, and that a material is free of truncated or enzymatically cleaved fragments, depends on HPLC and mass spectrometry, since each cleavage product has a predictable mass.

Why Peptidase Stability Matters in Assays

Fetal bovine serum and other serum supplements contain active peptidases, and many cultured cell types express membrane peptidases such as DPP-4, aminopeptidase N and neprilysin. A peptide added to a well may therefore be partly degraded before the readout, producing an apparent loss of potency. Heat inactivation of serum is intended to inactivate complement and should not be assumed to remove peptidase activity.

Several practices reduce the problem. Stability can be measured directly by incubating the peptide in the assay medium, sampling over time and quantifying the intact compound by HPLC or LC-MS; the masses of fragments that appear reveal where cleavage occurred, as outlined in our article on peptide stability and degradation. Serum-free conditions or shorter incubations limit exposure. Inhibitors can be added where they do not interfere with the biology under study: AEBSF or PMSF for serine peptidases, E-64 for cysteine peptidases, pepstatin A for aspartic peptidases, EDTA or 1,10-phenanthroline for metallopeptidases and bestatin for aminopeptidases. Each has caveats; EDTA strips metal ions that a signalling pathway may need, and AEBSF can covalently modify proteins.

Peptidases are also deliberate tools. Trypsin digestion underlies peptide mapping and bottom-up proteomics, and site-specific enzymes such as TEV protease remove affinity tags from recombinant proteins. When comparing results across lots, it helps to check the certificate of analysis and the methods summarised under peptide purity testing, so that degradation during an assay is not confused with impurities present from synthesis. These materials are for laboratory research use only and are not for human or veterinary use.

Peptidases at a Glance

Peptidases are classified by where they cut and by how they cut. Exopeptidases need a free terminus, endopeptidases cleave internally, and the serine, cysteine, aspartic and metallo mechanisms each leave characteristic sensitivities to inhibitors. The DPP-4 cleavage of GLP-1 shows how one enzyme can define a peptide’s lifetime and drive analogue design. In the laboratory, accounting for peptidase activity through stability checks, controls and suitable inhibitors is part of producing interpretable data.

References

1. Barrett AJ, Rawlings ND, Woessner JF (eds). Handbook of Proteolytic Enzymes. Academic Press.
2. Rawlings ND, Barrett AJ. MEROPS: the peptidase database. Nucleic Acids Research.
3. Schechter I, Berger A. On the size of the active site in proteases. I. Papain. Biochem. Biophys. Res. Commun. 1967, 27, 157–162.
4. Drucker DJ. The biology of incretin hormones. Cell Metabolism 2006, 3, 153–165.
5. Lau J, et al. Discovery of the once-weekly glucagon-like peptide-1 (GLP-1) analogue semaglutide. J. Med. Chem. 2015, 58, 7370–7380.