Regulation of a cytosolic and nuclear O-GlcNAc transferase -: Role of the tetratricopeptide repeats
Regulation of a cytosolic and nuclear O-GlcNAc transferase -: Role of the tetratricopeptide repeats
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DOI：
10.1074/jbc.274.45.32015
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发表时间：
1999-11-05
影响因子：
4.8
通讯作者：
Hart, GW
中科院分区：
文献类型：
--
作者：
Kreppel, LK;Hart, GW
O-GlcNAc转移酶(OGT)是一种独特的核糖基和胞质糖基转移酶，含有多个四肽重复序列。我们已经开始使用缺失分析和动力学研究相结合的方法来表征OGT的调控机制。在这里，我们证明了酶的p110亚基形成了同源和异源三聚体，这似乎与UDP-GlcNAc有不同的结合亲和力。OGT的多聚体结构域位于四肽重复结构域中，不是活性所必需的。对OGT的全长三聚体和截短单体形式的动力学分析表明，这两种形式都通过随机的双向动力学机制发挥作用。单体和三聚体对多肽底物具有相似的比活性和相似的K-m值。然而，它们与UDP-GlcNAc的结合亲和力不同，表明亚基相互作用影响酶活性。结果表明，重组OGT对UDP-GlcNAc有三个不同的K-m值，并且UDP-GlcNAc浓度调节OGT对多肽的亲和力，这表明OGT受到细胞核和细胞质中UDP-GlcNAc水平的精细调控。
The O-GlcNAc transferase (OGT) is a unique nuclear and cytosolic glycosyltransferase that contains multiple tetratricopeptide repeats. We have begun to characterize the mechanisms regulating OGT using a combination of deletion analysis and kinetic studies. Here we show that the p110 subunit of the enzyme forms both homo- and heterotrimers that appear to have different binding affinities for UDP-GlcNAc. The multimerization domain of OGT lies within the tetratricopeptide repeat domain and is not necessary for activity. Kinetic analyses of the full-length trimer and the truncated monomer forms of OGT suggest that both forms function through a random bi-bi kinetic mechanism. Both the monomer and trimer have similar specific activities and similar K-m values for peptide substrates. However, they differ in their binding affinities for UDP-GlcNAc, indicating that subunit interactions affect enzyme activity. The findings that recombinant OGT has three distinct K-m values for UDP-GlcNAc and that UDP-GlcNAc concentrations modulates the affinity of OGT for peptides suggest that OGT is exquisitely regulated by the levels of UDP-GlcNAc within the nucleus and cytoplasm.