C-terminal deletion of human tissue transglutaminase enhances magnesium-dependent GTP/ATPase activity.

The Journal of Biological Chemistry
T S LaiC S Greenberg

Abstract

Tissue transglutaminase (tTG) exhibits a magnesium-dependent GTP/ATPase activity that is involved in the regulation of the cell cycle and cell receptor signaling. The portion of the molecule involved in GTP/ATP hydrolysis is unknown. We expressed and purified a series of C-terminal truncation mutants of human tTG as glutathione S-transferase fusion proteins (DeltaS538, DeltaE447, DeltaP345, DeltaC290, DeltaV228, and DeltaF185) to determine the effect on GTP/ATPase activity. The truncation of the C terminus did not change significantly the apparent Km value for either GTP or ATP. In contrast, the Kcat value for GTP was increased by 4.6- and 3-fold for the DeltaS538 and DeltaE447 mutants, respectively. The DeltaP345 mutant had the highest hydrolysis activity with a 34-fold increase. The hydrolysis activity then declined to 8.1-, 8.7-, and 1. 9-fold for the DeltaC290, DeltaV228, and DeltaF185 mutants, respectively. The Kcat for ATP changed in parallel with the GTPase results. Thin layer chromatography analysis of the hydrolysis reaction products revealed that ATP was rapidly converted to ADP followed by a much slower conversion of ADP to AMP when incubated with wild type tTG or the DeltaP345 mutant. There was a substantial decreas...Continue Reading

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