Site-specific phosphorylation of L-form starch phosphorylase by the protein kinase activity from sweet potato roots.
A 78-amino acid insertion (L78) is found in the low-affinity type (L-form) of starch phosphorylase (L-SP, EC 2.4.1.1). This insertion blocks the starch-binding site on the L-SP molecule, and it decreases the binding affinity of L-SP toward starch. The computational analysis of the amino acid sequence on L78 predicts several phosphorylation sites at its Ser residues. Indeed, from the immunoblotting results using antibodies against phosphoamino acids, we observed that the purified L-SP from mature sweet potato (Ipomoea batatas) roots is phosphorylated. This observation led us to the detection of a protein kinase activity in the protein fraction of the crude extract from the sweet potato roots. The kinase was partially purified by liquid chromatography, and its native molecular mass was estimated as 338 kDa. An expressed peptide (L78P) containing the essential part of L78 was intensively phosphorylated by the kinase. However, H-SP (the high-affinity isomer of SP lacking the L78 insertion) and the proteolytic modified L-SP, which lost its L78 fragment, could not be phosphorylated. Furthermore, using L78P mutants by site-directed mutagenesis at Ser residues on L78, we demonstrate that only one Ser residue on L78 is phosphorylated by the kinase. These results imply that this kinase is specific to L-SP, or more precisely, to the L78 insertion. The in vitro phosphorylated L-SP shows higher sensitivity to proteolytic modification, but has no change in its kinetic parameters.
Young
Guang-Huar
GH
Department of Biochemical Science and Technology, Institute of Microbiology and Biochemistry, National Taiwan University, AC 2-520, Taipei 106, Taiwan.
Chen
Han-Min
HM
Lin
Chi-Tsai
CT
Tseng
Kuang-Ching
KC
Wu
Jiann-Shing
JS
Juang
Rong-Huay
RH
D000595:Amino Acid Sequence; D027723:Ipomoea batatas / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D018517:Plant Roots / Q000201:enzymology / Q000235:genetics; D011494:Protein Kinases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D020539:Sequence Analysis, Protein; D012694:Serine / Q000378:metabolism; D024941:Starch Phosphorylase / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity
D012694:Serine; D024941:Starch Phosphorylase; D011494:Protein Kinases