title: Continuous enzyme-coupled assay for microbial transglutaminase activity.
abstract: 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-ε-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.
authors.affiliation: Department of Chemistry, University of Ottawa, Ottawa, Ontario K1N 6H5, Canada.
authors.forename: Samuel K
authors.initials: SK
authors.lastname: Oteng-Pabi
authors.forename: Jeffrey W
authors.initials: JW
authors.lastname: Keillor
mesh_terms: D057075:Enzyme Assays / Q000379:methods*; D006877:Hydroxamic Acids / Q000737:chemistry / Q000378:metabolism; D057230:Limit of Detection; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D013302:Streptomyces / Q000201:enzymology*; D013379:Substrate Specificity; D011503:Transglutaminases / Q000032:analysis / Q000378:metabolism*
chemical_list: D006877:Hydroxamic Acids; D010455:Peptides; D011503:Transglutaminases
keywords: Enzyme-coupled assay; Glutamate dehydrogenase; Hydroxamate assay; Microbial transglutaminase; Microtiter plate; Transamidation