Transglutaminase 2 (TG2) is a multifunctional protein with diverse catalytic activities and biological roles. Its best studied function is the Ca2+-dependent transamidase activity leading to formation of γ-glutamyl-ε-lysine isopeptide crosslinks between proteins or γ-glutamyl-amine derivatives. TG2 has a poorly studied isopeptidase activity cleaving these bonds. We have developed and characterised TG2 mutants which are significantly deficient in transamidase activity while have normal or increased isopeptidase activity (W332F) and vice versa (W278F). The W332F mutation led to significant changes of both the Km and the Vmax kinetic parameters of the isopeptidase reaction of TG2 while its calcium and GTP sensitivity was similar to the wild type enzyme. The W278F mutation resulted in six times elevated amine incorporating transamidase activity demonstrating the regulatory significance of W278 and W332 in TG2 and that mutations can change opposed activities located at the same active site. The further application of our results in cellular systems may help to understand TG2 -driven physiological and pathological processes better and lead to novel therapeutic approaches where an increased amount of cross-linked proteins correlates with the manifestation of degenerative disorders.
Bibliographical noteThe final publication is available at Springer via http://dx.doi.org/10.1007/s00726-015-2063-5
- human transglutaminase 2
- isopeptidase activity
- regulation of activities
- moonlighting enzyme