title: The core domain of the tissue transglutaminase Gh hydrolyzes GTP and ATP.
abstract: Tissue transglutaminase (TGase II) catalyzes the posttranslational modification of proteins by transamidation of available glutamine residues and is also a guanosinetriphosphatase (GTPase) and adenosinetriphosphatase (ATPase). Based on its homology with factor XIIIA, an extracellular transglutaminase, the structure of TGase II is likely composed of an N-terminal beta-sandwich domain, an alpha/beta catalytic core, and two C-terminally located beta-barrels. Here we used a domain-deletion approach to identify the GTP and ATP hydrolytic domains of TGase II. Full-length TGase II and two domain-deletion mutants, one retaining the N-terminal beta-sandwich and core domains (betaSCore) and the other retaining only the core domain, were expressed as glutathione S-transferase (GST) fusion proteins and purified. GST-Full and GST-betaSCore exhibited calcium-dependent TGase activity, whereas GST-Core had no detectable TGase activity, indicating the beta-sandwich domain is required for TGase activity but the C-terminal beta-barrels are not. All three GST-TGase II fusion proteins were photoaffinity-labeled with [alpha-32P]-8-azidoGTP and were able to bind GTP-agarose. The GTPase activity of GST-betaSCore was equivalent to that of GST-Full, whereas the ATPase activity was approximately 40% higher than GST-Full. GST-Core had approximately 50% higher GTPase activity and approximately 75% higher ATPase activity than GST-Full. The GTPase and ATPase activities of each of the GST-TGase II fusion proteins were inhibited in a dose-dependent manner by both GTPgammaS and ATPgammaS. These results demonstrate that the GTP and ATP hydrolysis sites are localized within the core domain of TGase II and that neither the N-terminal beta-sandwich domain nor the C-terminal beta-barrels are required for either GTP or ATP hydrolysis. Taken together with previous work [Singh, U. S., Erickson, J. W., & Cerione, R. A. (1995) Biochemistry 34, 15863-15871; Lai, T.-S., Slaughter, T. F., Koropchak, C. M., Haroon, Z. A., & Greenberg, C. S. (1996) J. Biol. Chem. 271, 31191-31195] the results of this study indicate that the GTP and ATP hydrolysis sites are localized to a 5. 5 kDa (47 amino acid) region at the start of the core domain.
authors.affiliation: Victor Chang Cardiac Research Institute, St Vincent's Hospital, Darlinghurst, NSW, 2010, Australia. s.iismaa@victorchang.unsw.edu.au
authors.forename: S E
authors.initials: SE
authors.lastname: Iismaa
authors.forename: L
authors.initials: L
authors.lastname: Chung
authors.forename: M J
authors.initials: MJ
authors.lastname: Wu
authors.forename: D C
authors.initials: DC
authors.lastname: Teller
authors.forename: V C
authors.initials: VC
authors.lastname: Yee
authors.forename: R M
authors.initials: RM
authors.lastname: Graham
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D016208:Databases, Factual; D004926:Escherichia coli; D020558:GTP Phosphohydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019204:GTP-Binding Proteins; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011503:Transglutaminases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D000345:Affinity Labels; D011993:Recombinant Fusion Proteins; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases; D005982:Glutathione Transferase; D000251:Adenosine Triphosphatases; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins