Functional domain organization of the potato alpha-glucan, water dikinase (GWD): evidence for separate site catalysis as revealed by limited proteolysis and deletion mutants.
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.
Mikkelsen
René
R
Center for Molecular Plant Physiology (PlaCe), Plant Biochemistry Laboratory, The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Copenhagen, Denmark.
Blennow
Andreas
A
D017398:Alternative Splicing / Q000235:genetics; D001665:Binding Sites / Q000502:physiology; D020134:Catalytic Domain / Q000235:genetics / Q000502:physiology; D002942:Circular Dichroism / Q000379:methods; D019286:Endopeptidase K / Q000378:metabolism; D006868:Hydrolysis; D009154:Mutation / Q000235:genetics / Q000502:physiology; D010446:Peptide Fragments / Q000378:metabolism / Q000502:physiology; D010449:Peptide Mapping / Q000379:methods; D017866:Phosphotransferases (Paired Acceptors) / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000378:metabolism; D011198:Solanum tuberosum / Q000201:enzymology
D010446:Peptide Fragments; D011506:Proteins; D017866:Phosphotransferases (Paired Acceptors); C498198:alpha-glucan, water dikinase, Solanum tuberosum; D019286:Endopeptidase K