Official Full Name
Achromopeptidase
Background
Lysyl endopeptidase (EC 3.4.21.50, Achromobacter proteinase I, Achromobacter lyticus alkaline proteinase I, protease I, achromopeptidase, lysyl bond specific proteinase) is an enzyme. This enzyme catalyses the following chemical reaction:Preferential cleavage:Lys-, including-Lys-Pro-. This enzyme is isolated from Achromobacter lyticus.
Synonyms
Achromobacter proteinase I; Achromobacter lyticus alkaline proteinase I; protease I; achromopeptidase; lysyl bond specific proteinase; EC 3.4.21.50; 123175-82-6; Lysyl endopeptidase
Introductions
Achromopeptidase is a serine protease that specifically cleaves the C-terminal amino acid residues of lysine (including proline and glutamate), and is widely used in proteomics research, peptide sequencing, insulin preparation, identification of phosphorylated peptides, disulfide bond analysis, N-glycan analysis, etc. It has a great potential for commercial application, such as detection of glycated hemoglobin and cleaning of contact lenses.
Gene structure
Achromopeptidase is consists of signal peptide, pro-peptide, mature peptidase and exiension pepfide. The Achromopeptidase pro-peptide from P. ilyticaus and Lenzymogenesc were 653 and 670 amino acids, respectively, while the achromopeptidase pro-peptide from P. aeruginosa was only 462 amino acids.
Protein structure
Achromopeptidase contains three pairs of disulfide bonds, of which the disulfide bond C-S is the determinant of its optimal enzymatic activity and denaturant resistance under mechanical conditions < pH8.5 ~10.7). The catalytic triad of H57, D113 and S194 corresponds to the catalytic triad of H57, D102 and s195lR of trypsin, respectively.
Specificity
The substrate binding site of achromopeptidase is also known as S1 'specificity pocket.7 Its spatial geometry determines its high specificity, and TLCK is able to occupy the S1 specificity pocket instead of the peptide substrate, thus effectively inhibiting the enzymatic activity of ahromopeptidase. The substrate binding site of D225 forms a tight SI pocket with the neighboring T189, S214, which can only accommodate linear side chains. Among the amino acids, only the linear side chains of arginine and lysine are long enough to reach the bottom of the Sl pocket, but the fox group of the arginine side chain is spatially blocked from D225, preventing the binding of arginine to the Sl pocket. This unique geometric spatial structure explains the principle that it can only cleave the C-terminal amino acid residues of lysine exclusively.
Optimal pH
Neutral environment (pH 6.0~7.0) is the best condition for protease to convert the substrate into peptide fragments; the accumulation of WI69 and H210 at the catalytic center determines the optimal pH of achromopeptidase to be alkaline; therefore, mutation of this site becomes the key to solve the problem.
Self-degradation
Achromopeptidase is prone to autodegradation, which is an important factor limiting its application. For Protease IV, its self-degradation sites are K30, K48, K49, Kl55 and K203, which can produce 5kDa, 6kDa, 13kDa, 18kDa and 22kDa degradation fragments, respectively. Mutation or chemical modification of these lysine sites may reduce the tendency of self-degradation. The stability of achromopeptidase may be improved, but it may also bring about changes in the spatial structure and affect the enzymatic activity.
Applications
Proteomics is a discipline dedicated to the large-scale study of protein structure and function, and the complete enzymatic digestion of protein complexes is a modern high-resolution. High-throughput mass spectrometry is a key step in the identification and quantification of proteins. achromopeptidase is an essential tool for proteomics research.
Combined Lys-C and Lys-N digestion, followed by LC-MS/MS analysis, allows sequencing and identification of the N-terminal ends of different proteins in a mixture of proteins, including acetylated N-terminal ends, protein isomers, etc.
