The Core Domain of the Tissue Transglutaminase Gh Hydrolyzes GTP and ATP

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 trans- glutaminase, the structure of TGase II is likely composed of an N-terminal ‚-sandwich domain, an R/‚ catalytic core, and two C-terminally located ‚-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 ‚-sandwich and core domains (‚SCore) and the other retaining only the core domain, were expressed as glutathione S-transferase (GST) fusion proteins and purified. GST-Full and GST-‚SCore exhibited calcium-dependent TGase activity, whereas GST-Core had no detectable TGase activity, indicating the ‚-sandwich domain is required for TGase activity but the C-terminal ‚-barrels are not. All three GST-TGase II fusion proteins were photoaffinity-labeled with (R- 32 P)-8-azidoGTP and were able to bind GTP-agarose. The GTPase activity of GST-‚SCore was equivalent to that of GST- Full, whereas the ATPase activity was40% higher than GST-Full. GST-Core had50% higher GTPase activity and75% 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 GTPAS and ATPAS. 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 ‚-sandwich domain nor the C-terminal ‚-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.