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Biochemistry. 1997 Sep 30;36(39):11655-64.

The core domain of the tissue transglutaminase Gh hydrolyzes GTP and ATP.

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1
Victor Chang Cardiac Research Institute, St Vincent's Hospital, Darlinghurst, NSW, 2010, Australia. s.iismaa@victorchang.unsw.edu.au

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.

PMID:
9305955
DOI:
10.1021/bi970545e
[Indexed for MEDLINE]
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