6‐phosphofructokinase from Pichia pastoris: purification, kinetic and molecular characterization of the enzyme

Abstract

Abstract6-Phosphofructokinase from Pichia pastoris was purified for the first time to homogeneity applying seven steps, including pseudo-affinity dye-ligand chromatography on Procion Blue H-5R-Sepharose. The specific activity of the purified enzyme was about 80 U/mg. It behaves as a typically allosteric 6-phosphofructokinase exhibiting activation by AMP and fructose 2,6-bis(phosphate), inhibition by ATP and cooperativity to fructose 6-phosphate. However, in comparison with the enzymes from Saccharomyces cerevisiae and Kluyveromyces lactis, the activation ratio of 6-phosphofructokinase from Pichia pastoris by AMP is several times higher, the ATP inhibition is stronger and the apparent affinity to fructose 6-phosphate is significantly lower. Aqueous twophase affinity partitioning with Cibacron Blue F3G-A did not reflect remarkable structural differences of the nucleotide binding sites of the Pfks from Pichia pastoris and Saccharomyces cerevisiae. The structural organisation of the active enzyme seems to be different in comparison with hetero-octameric 6-phosphofructokinases from other yeast species. The enzyme was found to be a hetero-oligomer with an molecular mass of 975 kDa (sedimentation equilibrium measurements) consisting of two distinct types of subunits in an equimolar ratio with molecular masses of 113 kDa and 98 kDa (SDS-PAGE), respectively, and a third non-covalently complexed protein component (34 kDa, SDS-PAGE). The latter seems to be necessary for the catalytic activity of the enzyme. Sequencing of the N-terminus (VTKDSIXRDLEXENXGXXFF) and of peptide fragments by applying MALDI-TOF PSD, m/z 1517.3 (DAMNVVNH) and m/z 2177.2 [AQNCNVC(L/I)SVHEAHTM] gave no relevant information about the identity of this protein.

The dimorphic yeast Yarrowia lipolytica possesses an atypical phosphofructokinase: characterization of the enzyme and its encoding gene

“…In contrast to Pfk from other organisms, which are tetrameric proteins, the enzymes from yeasts are octameric, except for the Rhodotorula glutinis Pfk, which was suggested to be a tetramer (Schröter & Kopperschläger, 1996). However, the subunit composition of yeast Pfks varies between species: while the enzymes from S. cerevisiae, K. lactis, P. pastoris and C. albicans are heterooctameric proteins (Kopperschläger et al, 1977;Heinisch et al, 1993;Bär et al, 1997;Lorberg et al, 1999;Kirchberger et al, 2002), those of the fission yeasts Sch. pombe and Y. lipolytica are homo-octameric (Reuter et al, 2000;this work).…”

“…) is about one order of magnitude lower than those known for Pfks from other yeast species, such as S. cerevisiae (Hofmann & Kopperschläger, 1982), Schizosaccharomyces pombe (Reuter et al, 2000), P. pastoris (Kirchberger et al, 2002) and Kluyveromyces lactis (Bär et al, 1997).…”

A Novel Form of 6-Phosphofructokinase

“…Pfk activity measurement followed basically procedures described elsewhere (16). For kinetic studies a Pfk assay with simultaneous ATP and Fru 6-P regeneration (100 mM imidazole/HCl, pH 6.6, 100 mM KCl, 10 mM MgCl 2 , 20 mM potassium phosphate, 0.2 mM NADH ϩ , 0.6 mM phosphoenolpyruvate, 8.5 units of pyruvate kinase/ml, 7 units of lactate dehydrogenase/ml, 1 unit of fructose-1,6-bisphosphatase/ml; ATP, Fru 6-P, AMP, and Fru 2,6-P 2 as indicated) was used (16).…”

“…Pfk activity measurement followed basically procedures described elsewhere (16). For kinetic studies a Pfk assay with simultaneous ATP and Fru 6-P regeneration (100 mM imidazole/HCl, pH 6.6, 100 mM KCl, 10 mM MgCl 2 , 20 mM potassium phosphate, 0.2 mM NADH ϩ , 0.6 mM phosphoenolpyruvate, 8.5 units of pyruvate kinase/ml, 7 units of lactate dehydrogenase/ml, 1 unit of fructose-1,6-bisphosphatase/ml; ATP, Fru 6-P, AMP, and Fru 2,6-P 2 as indicated) was used (16). A two-state MonodWyman-Changeux model was applied to describe the ATP velocity curves under the assumptions: 1) an octameric allosteric mode, 2) AMP and Fru 2,6-P 2 binding to the R-state enzyme only, and 3) ATP serves as substrate (K S ATP ) in a hyperbolic manner, but acts also as allosteric inhibitor (K T ATP ),…”

“…Recently we co-purified a protein component together with the known Pfk ␣ and ␤ subunits (15)(16)(17) from the methylotrophic yeast Pichia pastoris. This unknown protein could only be separated under denaturating conditions and with loss of Pfk activity.…”