The starch-related R1 protein is an alpha -glucan, water dikinase.
To determine the enzymatic function of the starch-related R1 protein it was heterologously expressed in Escherichia coli and purified to apparent homogeneity. Incubation of the purified protein with various phosphate donor and acceptor molecules showed that R1 is capable of phosphorylating glucosyl residues of alpha-glucans at both the C-6 and the C-3 positions in a ratio similar to that occurring naturally in starch. Phosphorylation occurs in a dikinase-type reaction in which three substrates, an alpha-polyglucan, ATP, and H(2)O, are converted into three products, an alpha-polyglucan-P, AMP, and orthophosphate. The use of ATP radioactively labeled at either the gamma or beta positions showed that solely the beta phosphate is transferred to the alpha-glucan. The apparent K(m) of the R1 protein for ATP was calculated to be 0.23 microM and for amylopectin 1.7 mg x ml(-1). The velocity of in vitro phosphorylation strongly depends on the type of the glucan. Glycogen was an extremely poor substrate; however, the efficiency of phosphorylation strongly increased if the glucan chains of glycogen were elongated by phosphorylase. Mg(2+) ions proved to be essential for activity. Incubation of R1 with radioactively labeled ATP in the absence of an alpha-glucan showed that the protein phosphorylates itself with the beta, but not with the gamma phosphate. Autophosphorylation precedes the phosphate transfer to the glucan indicating a ping-pong reaction mechanism.
Ritte
Gerhard
G
Plant Physiology, Institute of Biochemistry and Biology, University of Potsdam, Karl-Liebknecht-Strasse 24-25 Haus 20, D-14476 Golm, Germany. ritte@rz.uni-potsdam.de
Lloyd
James R
JR
Eckermann
Nora
N
Rottmann
Antje
A
Kossmann
Jens
J
Steup
Martin
M
D000255:Adenosine Triphosphate; D000687:Amylopectin / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002384:Catalysis; D005936:Glucans / Q000378:metabolism*; D007700:Kinetics; D009004:Monosaccharide Transport Proteins; D010710:Phosphates / Q000378:metabolism; D010766:Phosphorylation; D017866:Phosphotransferases (Paired Acceptors) / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011198:Solanum tuberosum / Q000201:enzymology*; D013213:Starch / Q000378:metabolism
D000255:Adenosine Triphosphate; D000687:Amylopectin; D029681:Arabidopsis Proteins; D005936:Glucans; D009004:Monosaccharide Transport Proteins; D010710:Phosphates; D017866:Phosphotransferases (Paired Acceptors); D013213:Starch; C435922:At1g10760 protein, Arabidopsis; C022509:pyruvate, water dikinase