Jiang Peng, Pioszak Augen A, Ninfa Alexander J
Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109-0606, USA.
Biochemistry. 2007 Apr 3;46(13):4117-32. doi: 10.1021/bi0620508. Epub 2007 Mar 14.
Glutamine synthetase adenylyltransferase (ATase) regulates the activity of glutamine synthetase by adenylylation and deadenylylation in response to signals of nitrogen and carbon status: glutamine, alpha-ketoglutarate, and the uridylylated and unmodified forms of the PII signal transduction protein. ATase consists of two conserved nucleotidyltransferase (NT) domains linked by a central region of approximately 200 amino acids. Here, we study the activities and regulation of mutated and truncated forms of ATase. Our results indicate the following. (i) The N-terminal NT domain contained the adenylyl-removing (AR) active site, and the C-terminal NT domain contained the adenylyltransferase (AT) active site. (ii) The enzyme contained a glutamine binding site, and glutamine increased the affinity for PII. (iii) The enzyme appeared to contain multiple sites for the binding of PII and PII-UMP. (iv) Truncated versions of ATase missing the C-terminal (NT) domain lacked both AT and AR activity, suggesting a role for the C-terminal NT domain in both activities. (v) The purified C-terminal NT domain and larger polypeptides containing this domain had significant basal AT activity, which was stimulated by glutamine. These polypeptides were indifferent to PII and PII-UMP, or their ATase activity was inhibited by either PII or PII-UMP. (vi) Certain point mutations in the central region or an internal deletion removing most of this part of the protein eliminated the AR activity and eliminated activation of the AT activity by PII, while not eliminating the binding of PII or PII-UMP. That is, these mutations in the central region appeared to destroy the communication between the PII and PII-UMP binding sites and the AT and AR active sites. (vii) Certain mutations in the central region of ATase appeared to dramatically improve the binding of glutamine to the enzyme. (viii) While the isolated AT and AR domains of ATase bound poorly to PII and PII-UMP, these domains bound PII and PII-UMP significantly better when linked to the central region of ATase. Together, our results indicate a highly coordinated enzyme, in which the AT and AR domains participate in each other's regulation and distant regulatory sites are in communication with each other. A model for the regulation of ATase by glutamine, PII, and PII-UMP consistent with all data is presented.
谷氨酰胺合成酶腺苷酰转移酶（ATase）通过腺苷酸化和去腺苷酸化作用，响应氮和碳状态信号（谷氨酰胺、α-酮戊二酸以及PII信号转导蛋白的尿苷酰化和未修饰形式）来调节谷氨酰胺合成酶的活性。ATase由两个保守的核苷酸转移酶（NT）结构域组成，中间由大约200个氨基酸的区域相连。在此，我们研究了ATase突变体和截短形式的活性及调控。我们的结果表明如下几点：（i）N端NT结构域包含去腺苷酰化（AR）活性位点，C端NT结构域包含腺苷酰转移酶（AT）活性位点。（ii）该酶含有一个谷氨酰胺结合位点，谷氨酰胺可增加其对PII的亲和力。（iii）该酶似乎含有多个PII和PII-UMP结合位点。（iv）缺失C端（NT）结构域的ATase截短形式同时缺乏AT和AR活性，这表明C端NT结构域在这两种活性中均发挥作用。（v）纯化的C端NT结构域以及包含该结构域更大的多肽具有显著的基础AT活性，谷氨酰胺可刺激该活性。这些多肽对PII和PII-UMP不敏感，或者它们的ATase活性被PII或PII-UMP抑制。（vi）中央区域的某些点突变或去除该蛋白大部分此区域的内部缺失消除了AR活性，并消除了PII对AT活性的激活作用，同时并未消除PII或PII-UMP的结合。也就是说，中央区域的这些突变似乎破坏了PII和PII-UMP结合位点与AT和AR活性位点之间的通讯。（vii）ATase中央区域的某些突变似乎显著改善了谷氨酰胺与该酶的结合。（viii）虽然ATase分离的AT和AR结构域与PII和PII-UMP结合不佳，但当与ATase的中央区域相连时，这些结构域与PII和PII-UMP的结合明显更好。总之，我们的结果表明这是一种高度协调的酶，其中AT和AR结构域相互参与彼此的调控，且远距离调控位点相互通讯。本文提出了一个与所有数据一致的谷氨酰胺、PII和PII-UMP对ATase调控的模型。
