Lai T S, Slaughter T F, Koropchak C M, Haroon Z A, Greenberg C S
Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA.
J Biol Chem. 1996 Dec 6;271(49):31191-5. doi: 10.1074/jbc.271.49.31191.
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 decrease in the calcium-dependent TGase activity when the last 149 amino acid residues were deleted from the C terminus. Less than 5% of the TGase activity was detected for the DeltaS538 and DeltaE447 mutants. In conclusion, we have located the ATP and GTP hydrolytic domain to amino acid residues 1-185. The C terminus functions to inhibit the expression of endogenous GTP/ATPase activity of tTG, and the potential role of the C terminus in modulating this activity is discussed.
组织转谷氨酰胺酶（tTG）具有镁依赖性GTP/ATP酶活性，参与细胞周期和细胞受体信号传导的调节。分子中参与GTP/ATP水解的部分尚不清楚。我们表达并纯化了一系列人tTG的C末端截短突变体，作为谷胱甘肽S-转移酶融合蛋白（DeltaS538、DeltaE447、DeltaP345、DeltaC290、DeltaV228和DeltaF185），以确定对GTP/ATP酶活性的影响。C末端的截短对GTP或ATP的表观Km值没有显著改变。相比之下，DeltaS538和DeltaE447突变体的GTP的Kcat值分别增加了4.6倍和3倍。DeltaP345突变体具有最高的水解活性，增加了34倍。然后，DeltaC290、DeltaV228和DeltaF185突变体的水解活性分别下降到8.1倍、8.7倍和1.9倍。ATP的Kcat与GTP酶结果平行变化。水解反应产物的薄层色谱分析表明，当与野生型tTG或DeltaP345突变体一起孵育时，ATP迅速转化为ADP，随后ADP转化为AMP的速度要慢得多。当从C末端删除最后149个氨基酸残基时，钙依赖性转谷氨酰胺酶活性大幅下降。DeltaS538和DeltaE447突变体检测到的转谷氨酰胺酶活性不到5%。总之，我们已将ATP和GTP水解结构域定位到氨基酸残基1-185。C末端起到抑制tTG内源性GTP/ATP酶活性表达的作用，并讨论了C末端在调节该活性中的潜在作用。
