The Core Domain of the Tissue Transglutaminase G h Hydrolyzes GTP and ATP † Siiri E. Iismaa,* ,‡ Liping Chung, ‡ Ming-Jie Wu, ‡ David C. Teller, § Vivien C. Yee, § and Robert M. Graham ‡ Victor Chang Cardiac Research Institute, St Vincent’s Hospital, Darlinghurst, NSW, 2010, Australia, and Department of Biochemistry, UniVersity of Washington, Seattle, Washington 98195 ReceiVed March 10, 1997; ReVised Manuscript ReceiVed July 7, 1997 X 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 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 was ∼40% higher than GST-Full. GST-Core had ∼50% higher GTPase activity and ∼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 GTPγS and ATPγS. 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. The GTP-binding protein (G-protein), 1 G h , is a dual function guanosinetriphosphatase (GTPase)/transglutaminase. G h was identified as a novel 74 kDa protein that couples to the R 1 -adrenergic receptor in rat liver plasma membranes and mediates R 1 -transmembrane signaling by stimulating a membrane-bound phospholipase C (Im & Graham, 1990; Im et al., 1990, 1992). Similar coupling of G h with R 1 - adrenergic receptors has been demonstrated in rat, dog, rabbit, bovine, and human heart preparations (Baek et al., 1993; Braun & Walsh, 1993). The molecular mass of the G h family varies slightly in different species, ranging from M r ’s of 74 to 80 kDa (Baek et al., 1993; Braun & Walsh, 1993). G h has been shown to mediate phospholipase C activation by R 1B - and R 1D - but not R 1A -adrenergic receptors (Nakaoka et al., 1994; Chen et al., 1996) and appears to couple also to oxytocin receptors (Baek et al., 1996). Partial amino acid sequence analysis and immunological characterization (Na- kaoka et al., 1994) have identified G h as a tissue trans- glutaminase type II (TGase II, R-glutaminyl peptide:amine γ-glutamyltransferase, EC 2.3.2.13). A GTP-binding protein in rabbit liver nuclear membranes has also recently been identified as TGase II (Singh et al., 1995). TGases are Ca 2+ -dependent acyl transferases that catalyze the formation of an amide bond between the γ-carboxamide groups of peptide-bound glutamine residues and the primary amino groups in various compounds, including the ǫ-amino group of lysines in certain proteins (Folk, 1980). The TGases are a family of closely related thiol enzymes that are derived from a common ancestor (Aeschlimann & Paulsson, 1994). Five enzymatically active TGases have been identified [see Greenberg et al. (1991) and Aeschlimann and Paulsson (1994) for reviews]. These include keratinocyte TGase (TGase I), which is primarily membrane-associated and plays a major role in the formation of the cornified cell envelope of the epidermis; tissue TGase (TGase II, G h ), which is ubiquitously expressed in mammalian tissues, both in membrane and in cytosolic fractions, and has been implicated in apoptosis, cell adhesion, and signal transduction (Nakaoka et al., 1994); epidermal TGase (TGase III), a soluble proenzyme involved in differentiating epidermal and hair follicle cells that requires proteolytic activation; prostate TGase (TGase IV), which in rodents is involved in the formation of copulatory plugs in the female genital tract after † This work was supported by an Eccles Award and a project grant (951001) from the National Health and Medical Research Council, Australia, and National Institutes of Health Grant HL-50355. * To whom correspondence should be addressed: Tel, 61-2-9295- 8523; Fax, 61-2-9295-8501; e-mail, s.iismaa@victorchang.unsw.edu.au. ‡ Victor Chang Cardiac Research Institute. § University of Washington. X Abstract published in AdVance ACS Abstracts, September 1, 1997. 1 Abbreviations: ATP, adenosine 5′-triphosphate; G-protein, GTP- binding protein; GST, glutathione S-transferase; GST-Full, GST fused to full-length TGase II; GST-SCore, -sandwich and core domains of TGase II fused to GST; GST-Core, core domain of TGase II fused to GST; GTP, guanosine 5′-triphosphate; IPTG, isopropyl 1-thio--D- galactopyranoside; SDS-PAGE, sodium dodecyl sulfate-polyacryla- mide gel electrophoresis; TGase II, tissue transglutaminase type II. 11655 Biochemistry 1997, 36, 11655-11664 S0006-2960(97)00545-X CCC: $14.00 © 1997 American Chemical Society