Purification, microsequencing and cloning of spinach ATP-dependent phosphofructokinase link sequence and function for the plant enzyme

Abstract

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 Km value for ATP of 81 μm. 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. © 2007 The Authors.