tailieunhanh - Báo cáo khoa học: 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 ) 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 mUÆmg )1 and aKmvalue for ATP of 81 lm. | ỊFEBS Journal Purification microsequencing and cloning of spinach ATP-dependent phosphofructokinase link sequence and function for the plant enzyme Christian Winkler Britta Delvos William Martin and Katrin Henze Institute of Botany III University of Dusseldorf Germany Keywords ATP-PFK sequence subunits Correspondence K. Henze Institute of Botany III University of Dusseldorf D-40225 Dusseldorf Germany Fax 49 211 811 3554 Tel 49 211 811 2339 E-mail winklech@ Database The sequences reported here have been submitted to the GenBank database under the accession numbers DQ437575 and DQ437576 Received 5 October 2006 revised 7 November 2006 accepted 10 November 2006 Despite its importance in plant metabolism no sequences of higher plant ATP-dependent phosphofructokinase EC 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 pM. The purified enzyme was not activated by phosphate but slightly inhibited instead suggesting that it was the chloroplast isoform. The inclusion of adenosine 5 - P y-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. doi Phosphofructokinase PFK catalyzes the phosphorylation of D-fructose 6-phosphate to D-fructose 1 6-bis-phosphate. The enzyme has been .