General · EdgepediaSeptember 17, 20267 min read

Lysyl endopeptidase

Endoproteinase Lys-C (Lys-C, lysyl endopeptidase) is a lysine-specific serine endoprotease secreted by the Gram-negative soil bacterium Lysobacter enzymogenes that hydrolyzes peptide bonds on the carboxyl side of lysine residues, including the Lys-Pro bond. It is classified as EC 3.4.21.50 and is sold under the name Endoproteinase Lys-C by Roche and other suppliers for protein sequencing and proteomics sample preparation.¹^•²

Key fact	Detail
Source and class	Serine endoprotease from Lysobacter enzymogenes; EC 3.4.21.50; peptidase family S1 (trypsin-like)¹^•³
Cleavage rule	Preferential cleavage Lys-\|-Xaa, including Lys-\|-Pro; Lys-Glu bonds are also cleaved, with some minor non-specific cleavage reported¹^•²
Denaturant tolerance	Remains active in 8 M urea, 2 M guanidine-HCl, 1% SDS, 2% CHAPS, and 40% acetonitrile; maximal activity at pH 8⁴
Molecular mass	About 30 kDa (lyophilized MS-grade enzyme); 33 kDa by reducing SDS-PAGE for the Roche sequencing-grade product⁴^•⁵
Typical use	Sequential Lys-C then trypsin digestion in bottom-up proteomics; in a 293T cell study this raised identified proteins from 2,070 to 2,317 (an 11.9% increase)⁶
Working conditions	Enzyme-to-protein ratios of 1:20 to 1:100 (w/w); incubation 2 to 18 hours at 37 °C²
Inhibitors	Aprotinin, DFP, leupeptin, and TLCK⁷

Enzymology

Lys-C is a trypsin-type serine protease with a high pH optimum that hydrolyzes Lys-Xaa peptide bonds.⁸ In ammonium bicarbonate or Tris-HCl buffers at pH 8.5 it cleaves specifically at the carboxyl side of lysine, producing a limited number of fragments, which is the property that made it useful for protein sequencing work.² The Roche sequencing-grade preparation has an optimum pH of 8.5 to 8.8 and a specific activity of at least 200 units/mg protein.⁵ Unlike trypsin, Lys-C cleaves lysines followed by prolines, and its prototypic peptides typically carry higher charge states.⁴

Its most distinctive practical property is stability under strongly denaturing conditions. Lys-C retains most of its activity in 2.0 M urea or 0.1% SDS, and MS-grade preparations remain active in 8 M urea, 2 M guanidine-HCl, 1% SDS, 2% CHAPS, and 40% acetonitrile.²^•⁴ This allows digestion directly on denatured, unfolded protein without prior desalting or buffer exchange, which is why Lys-C is often the first enzyme applied in a proteomics workflow.⁹

Structure

The crystal structures of wild-type Lys-C and its K30R variant, covalently inhibited by N(α)-p-tosyl-lysyl chloromethylketone (TLCK), were determined to 1.1 and 0.9 Å resolution (PDB entries 4NSV and 4NSY), giving unusually precise views of the active and substrate-binding sites for this enzyme subfamily.⁸^•³ The structures show a trypsin-like fold with a catalytic triad at His57, Asp113, and Ser194 (LepA numbering for the homologous lysyl endopeptidase), and lysine specificity conferred by Asp225.¹⁰ The high pH optimum is attributed to electrostatic effects of an aromatic Tyr/His stack on the catalytic aspartate, a feature shared across this enzyme subfamily. Unexpectedly, the electron density indicates that a hydroxyl group, rather than the expected oxyanion, binds in the partially solvent-exposed oxyanion hole, suggesting that protonation of the alkoxide catalytic intermediate may recur during serine protease catalysis.⁸

History

The enzyme's value for primary structure determination was described by Peter A. Jekel, Wicher J. Weijer, and Jaap J. Beintema of the University of Groningen in Analytical Biochemistry in 1983, in a paper titled "Use of endoproteinase Lys-C from Lysobacter enzymogenes in protein sequence analysis"; the enzyme was already commercially available at that time.¹¹ Lysobacter enzymogenes also secretes related proteases, including α-lytic protease, a chymotrypsin-like bacterial serine protease, and an arginyl endopeptidase (endoproteinase Arg-C) whose gene was cloned in 1998.¹²

Use in proteomics

In bottom-up mass spectrometry proteomics, proteins are digested into peptides for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Trypsin is the standard protease because it generates peptides with basic C-terminal residues, but it cleaves less efficiently after lysine than after arginine, particularly when the lysine is followed by proline or flanked by acidic residues, producing missed cleavages.⁶^•⁴

Sequential digestion. The common remedy is Lys-C/trypsin sequential digestion: the protein is first digested with Lys-C while still dissolved in high urea, then the urea is diluted and trypsin is added. In the protocol used by Qian and colleagues on a 293T human renal epithelial cell extract, the initial Lys-C digest ran 3 hours at a 1:100 enzyme-to-protein ratio (w:w) in 8 M urea, after which the sample was diluted to 2 M urea for a 16-hour trypsin digest at 1:50, all in 100 mM ammonium bicarbonate, pH 8.0, at 37 °C.⁶ The tandem digest identified 2,317 proteins against 2,070 for trypsin alone, an 11.9% increase, and reduced missed cleavage sites, especially at lysine (K) sites, while raising average sequence coverage.⁶ Commercial trypsin/Lys-C mixes are sold for the same purpose, since trypsin alone does not fully digest proteins at the carboxyl end of lysine and arginine residues.⁴

Other applications. Because Lys-C produces longer peptides than trypsin, it serves as an alternative protease for increased peptide length, multiple-protease digestion strategies, and oxygen-18 labeling.¹³ In biotherapeutics, Lys-C peptide mapping is used for sequence confirmation and structural characterization of antibodies, including disulfide bond mapping; digestion under neutral pH with methionine as a scavenger minimizes deamidation and oxidation artifacts, and optimized methods have achieved 100% sequence coverage for a monoclonal antibody.¹⁴^•⁹ Lys-C digestion is also used in phosphopeptide enrichment workflows because it generates peptides with primary amines at both termini, allowing double labeling with amine-reactive isobaric tags.⁴

Related enzymes and variants

Lysine-specific peptidases related to Lys-C occur in other bacteria: lysyl endopeptidases have been found in the culture broths of Achromobacter lyticus, Lysobacter enzymogenes, and Pseudomonas aeruginosa, and a second lysine-specific serine protease (LepB) was identified in Lysobacter sp. strain IB-9374.¹⁰ Peptidyl-Lys metalloendopeptidases (PKMs), which cleave at lysine residues in the P1 position, function as complementary enzymes to Lys-C and can be combined with it to distinguish protein N- and C-termini in a single experiment.¹⁵ On the arginine side, a combined Lys-C/Arg-C Ultra protocol for data-independent acquisition proteomics achieved more than 99% arginine cleavage efficiency and 95% lysine cleavage efficiency, cutting arginine missed cleavages from about 30% to under 1% relative to a standard Lys-C/trypsin protocol.¹⁶

Handling

The lyophilized sequencing-grade enzyme is stored desiccated at 2 to 8 °C and reconstituted in water; after reconstitution, aliquots can be frozen for several weeks, and stock solutions are stable for up to 2 days at 4 °C or up to 1 month at −20 °C.²^•⁷ Self-digestion may occur at temperatures above 37 °C, and a standard peptide such as melittin or oxidized insulin B chain is recommended as a control substrate.²

References

Initially written Sep 17, 2026 · Reviewed: Oct 21, 2026 · Edited: Oct 11, 2026; Oct 15, 2026; Oct 21, 2026