Continuous enzyme-coupled assay for microbial transglutaminase activity
| Autor: | Samuel K. Oteng-Pabi, Jeffrey W. Keillor |
|---|---|
| Rok vydání: | 2013 |
| Předmět: |
Tissue transglutaminase
Biophysics Peptide Hydroxamic Acids Biochemistry Substrate Specificity 03 medical and health sciences 0302 clinical medicine Limit of Detection Molecular Biology 030304 developmental biology Enzyme Assays chemistry.chemical_classification 0303 health sciences Isopeptide bond Bioconjugation Transglutaminases biology Glutamate dehydrogenase Substrate (chemistry) Cell Biology Streptomyces Amino acid Glutamine chemistry 030220 oncology & carcinogenesis biology.protein Peptides |
| Zdroj: | Analytical biochemistry. 441(2) |
| ISSN: | 1096-0309 |
| Popis: | Transglutaminases (protein-glutamine:amine γ-glutamyltransferase, EC 2.3.2.13) are a family of calcium-dependent enzymes that catalyze an acyl transfer between glutamine residues and a wide variety of primary amines. When a lysine residue acts as the acyl-acceptor substrate, a γ-glutamyl-e-lysine isopeptide bond is formed. This isopeptide bond formation represents protein cross-linking, which is critical to several biological processes. Microbial transglutaminase (mTG) is a bacterial variant of the transglutaminase family, distinct by virtue of its calcium-independent catalysis of the isopeptidic bond formation. Furthermore, mTG's promiscuity in acyl-acceptor substrate preference highlights its biocatalytic potential. The acyl-donor substrate, however, is limited in its scope; the amino acid sequences flanking glutamine residues dramatically affect substrate specificity and activity. Here, we have developed and optimized a modified glutamate dehydrogenase assay with the intention of analyzing potential high-affinity peptides. This direct continuous assay presents significant advantages over the commonly used hydroxamate assay, including generality, sensitivity, and ease of manipulation. Furthermore, we identified 7M48 (WALQRPH), a high-affinity peptide that shows greater affinity with mTG (K(M)=3 mM) than the commonly used Cbz-Gln-Gly (K(M)=58 mM), attesting to its potential for application in biocatalysis and bioconjugation. |
| Databáze: | OpenAIRE |
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