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Purification, microsequencing and cloning of spinach ATP‐dependent phosphofructokinase link sequence and function for the plant enzyme
Author(s) -
Winkler Christian,
Delvos Britta,
Martin William,
Henze Katrin
Publication year - 2007
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2006.05590.x
Subject(s) - homotetramer , phosphofructokinase , biochemistry , spinach , enzyme , biology , pyrophosphate , phosphofructokinase 2 , complementary dna , peptide sequence , adenosine triphosphate , microbiology and biotechnology , glycolysis , protein subunit , gene
Despite its importance in plant metabolism, no sequences of higher plant ATP‐dependent phosphofructokinase (EC 2.7.1.11) are annotated in the databases. We have purified the enzyme from spinach leaves 309‐fold to electrophoretic homogeneity. The purified enzyme was a homotetramer of ∼52 kDa subunits with a specific activity of 600 mU·mg −1 and a K m value for ATP of 81 µ m . The purified enzyme was not activated by phosphate, but slightly inhibited instead, suggesting that it was the chloroplast isoform. The inclusion of adenosine 5′‐(β,γ‐imido)triphosphate was conducive to enzyme activitiy during the purification protocol. The sequences of eight tryptic peptides from the final protein preparation, which did not utilize pyrophosphate as a phosphoryl donor, were determined and an exactly corresponding cDNA was cloned. The sequence of enzymatically active spinach ATP‐dependent phosphofructokinase suggests that a large family of genomics‐derived higher plant sequences currently annotated in the databases as putative pyrophosphate‐dependent phosphofructokinases according to sequence similarity is misannotated with respect to the cosubstrate.