title: Isopeptidase activity of human transglutaminase 2: disconnection from transamidation and characterization by kinetic parameters.
abstract: Transglutaminase 2 (TG2) is a multifunctional protein with diverse catalytic activities and biological roles. Its best studied function is the Ca(2+)-dependent transamidase activity leading to formation of γ-glutamyl-ε-lysine isopeptide crosslinks between proteins and 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 K m and the V max 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 crosslinked proteins correlates with the manifestation of degenerative disorders.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Debrecen, Egyetem tér 1., Debrecen, 4012, Hungary.
authors.forename: Róbert
authors.initials: R
authors.lastname: Király
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Debrecen, Egyetem tér 1., Debrecen, 4012, Hungary.
authors.forename: Kiruphagaran
authors.initials: K
authors.lastname: Thangaraju
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Debrecen, Egyetem tér 1., Debrecen, 4012, Hungary.
authors.forename: Zsófia
authors.initials: Z
authors.lastname: Nagy
authors.affiliation: School of Life and Health Sciences, Aston University, Birmingham, UK.
authors.forename: Russell
authors.initials: R
authors.lastname: Collighan
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Debrecen, Egyetem tér 1., Debrecen, 4012, Hungary.
authors.forename: Zoltán
authors.initials: Z
authors.lastname: Nemes
authors.affiliation: School of Life and Health Sciences, Aston University, Birmingham, UK.
authors.forename: Martin
authors.initials: M
authors.lastname: Griffin
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Debrecen, Egyetem tér 1., Debrecen, 4012, Hungary. fesus@med.unideb.hu.|MTA-DE Stem Cell, Apoptosis and Genomics Research Group of Hungarian Academy of Sciences, Faculty of Medicine, University of Debrecen, Egyetem tér 1., Debrecen, 4012, Hungary. fesus@med.unideb.hu.
authors.forename: László
authors.initials: L
authors.lastname: Fésüs
mesh_terms: D000588:Amines / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D019759:Carbon-Nitrogen Lyases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D020125:Mutation, Missense; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D000588:Amines; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases; D019204:GTP-Binding Proteins; D019759:Carbon-Nitrogen Lyases; C034300:isopeptidase; D002118:Calcium
keywords: Human transglutaminase 2; Isopeptidase activity; Moonlighting enzyme; Regulation of activities; Transamidation; γ-Glutamyl-hydrolase