Purification, microsequencing and cloning of spinach ATP-dependent phosphofructokinase link sequence and function for the plant enzyme.
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 approximately 52 kDa subunits with a specific activity of 600 mU x mg(-1) and a Km value for ATP of 81 microm. The purified enzyme was not activated by phosphate, but slightly inhibited instead, suggesting that it was the chloroplast isoform. The inclusion of adenosine 5'-(beta,gamma-imido)triphosphate was conducive to enzyme activity 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.
Winkler
Christian
C
Institute of Botany III, University of Düsseldorf, Germany. winklech@uni-duesseldorf.de
Delvos
Britta
B
Martin
William
W
Henze
Katrin
K
D000595:Amino Acid Sequence; D002736:Chloroplasts / Q000378:metabolism; D003001:Cloning, Molecular; D019281:Dimerization; D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D025481:Phosphofructokinase-2 / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D010802:Phylogeny; D017434:Protein Structure, Tertiary; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D018724:Spinacia oleracea / Q000201:enzymology*; D014357:Trypsin / Q000737:chemistry / Q000494:pharmacology
D010455:Peptides; D025481:Phosphofructokinase-2; D014357:Trypsin