Tissue transglutaminase (tTG) catalyzes a Ca2؉ -dependent transglutaminase (TGase) activity that stabilizes tissues and a GTP hydrolysis activity that regulates cell receptor signaling. The purpose of this study was to examine the true substrates for nucleotide hydrolysis and the effects of these substrates on modulating the dual enzymatic activities of tTG. We found that Mg-GTP and Mg-ATP are the true substrates of the hydrolysis reaction. tTG hydrolyzed Mg-GTP and Mg-ATP at similar rates and interacted with Mg-ATP (K m ؍ 38 ؎ 10 M) at a 3-fold greater steady-state affinity than with Mg-GTP (K m ؍ 130 ؎ 35 M). In addition, Mg-ATP inhibited GTP hydrolysis (IC 50 ؍ 24 M), whereas 1 mM Mg-GTP reduced ATP hydrolysis by only 20%. Furthermore, the TGase activity of tTG was inhibited by Mg-GTP, Mg-GDP, and Mg-GMP, with IC 50 values of 9, 9, and 400 M, respectively, whereas the Mg-adenine nucleotides were ineffective. Kinetic analysis of the hydrolysis reaction demonstrates the presence of separate binding sites for Mg-GTP and Mg-ATP. Finally, we found that Mg-GTP protected tTG from proteolytic degradation by trypsin, whereas Mg-ATP was ineffective. In conclusion, we report that Mg-GTP and Mg-ATP can bind to distinct sites and serve as substrates for nucleotide hydrolysis. Furthermore, binding of Mg-GTP causes a conformational change and the inhibition of TGase activity, whereas Mg-ATP is ineffective. The implication of these findings in regulating the intracellular and extracellular function of tTG is discussed.Tissue transglutaminase (tTG) 1 exhibits two distinct enzymatic activities (1, 2): a calcium-dependent transglutaminase (TGase) activity that plays an important role in protein crosslinking and the regulation of apoptosis, cell morphology, cell adhesion, and tumor growth and metastasis (1-7) and a GTP binding and hydrolysis activity that is involved in signal transduction and that plays a role in cell cycle progression (8, 9). The biochemical factors modulating these divergent activities remain poorly defined.The TGase activity function requires the active-site cysteine 277, whereas the GTPase function does not, suggesting the presence of different catalytic sites (10). A putative calciumbinding site in human tTG required for TGase activity is located between Ser-430 and His-441 (11). Magnesium ions are required for GTP and ATP hydrolysis (10), and the location of this site(s) is unknown.We previously reported that GTP was reversibly bound to guinea pig tTG and inhibited TGase activity by inducing a conformational change that could be reversed by calcium ions (12). A single GTP-binding site was subsequently reported for human erythrocyte tTG, and this binding caused a reduction in affinity for calcium ions (13,14). Recent studies from our laboratory demonstrated that the GTP-and ATP-binding domains are located in the N-terminal 185 amino acid residues (15).Tissue transglutaminase is found in several distinct compartments in cells and tissues. Inside cells, tTG appears in the cytoplasm, although a small fr...