Functional domain organization of the potato α-glucan, water dikinase (GWD):: evidence for separate site catalysis as revealed by limited proteolysis and deletion mutants
Functional domain organization of the potato α-glucan, water dikinase (GWD):: evidence for separate site catalysis as revealed by limited proteolysis and deletion mutants
复制标题
DOI：
10.1042/bj20041119
复制
发表时间：
2005-01-15
影响因子：
4.1
通讯作者：
Blennow, A
中科院分区：
文献类型：
--
作者：
Mikkelsen, R;Blennow, A
马铃薯块茎（Solanum tuberosum）GWD（α-葡聚糖，水二激酶）通过二激酶型反应机制催化淀粉的磷酸化，其中 ATP 的 β-磷酸转移到支链淀粉的葡萄糖基残基上。 GWD 显示与细菌丙酮酸、水二激酶和 PPDK（丙酮酸、磷酸二激酶）的序列相似性。在本研究中，我们研究了 GWD 的结构与功能关系。对 GWD 蛋白水解片段的分析，结合肽微测序和缺失突变体的生成，表明 GWD 由 37、24、21、36 和 38 kDa 的 5 个离散结构域组成。催化组氨酸介导磷酰基从 ATP 转移到淀粉，位于 36 kDa 片段上，而 38 kDa C 末端片段包含 ATP 结合位点。葡聚糖分子的结合似乎仅限于包含三个 N 末端结构域的区域。生成缺失突变体是为了研究假定的 ATP 和葡聚糖结合域的功能相互依赖性。表达 36 和 38 kDa C 端结构域的 GWD 的截短形式被发现可以催化 E + ATP --> E-P + AMP + P-i（其中 P-i 代表正磷酸盐）部分反应，但不能催化 E-P + 葡聚糖 --> E + 葡聚糖-P 部分反应。 CD 实验为 GWD 自身磷酸化的大结构变化提供了证据，表明 GWD 采用与 PPDK 相似的旋转结构域机制进行酶促磷酸转移。
The potato tuber (Solanum tuberosum) GWD (alpha-glucan, water dikinase) catalyses the phosphorylation of starch by a dikinase-type reaction mechanism in which the beta-phosphate of ATP is transferred to the glucosyl residue of amylopectin. GWD shows sequence similarity to bacterial pyruvate, water dikinase and PPDK (pyruvate, phosphate dikinase). In the present study, we examine the structure-function relationship of GWD. Analysis of proteolytic fragments of GWD, in conjunction with peptide microsequencing and the generation of deletion mutants, indicates that GWD is comprised of five discrete domains of 37, 24, 21, 36 and 38 kDa. The catalytic histidine, which mediates the phosphoryl group transfer from ATP to starch, is located on the 36 kDa fragment, whereas the 38 kDa C-terminal fragment contains the ATP-binding site. Binding of the glucan molecule appears to be confined to regions containing the three N-terminal domains. Deletion mutants were generated to investigate the functional interdependency of the putative ATP- and glucan-binding domains. A truncated form of GWD expressing the 36 and 38 kDa C-terminal domains was found to catalyse the E + ATP --> E-P + AMP + P-i (where P-i stands for orthophosphate) partial reaction, but not the E-P + glucan --> E + glucan-P partial reaction. CD experiments provided evidence for large structural changes on autophosphorylation of GWD, indicating that GWD employs a swivelling-domain mechanism for enzymic phosphotransfer similar to that seen for PPDK.