6-phosphofructokinase from Pichia pastoris: purification, kinetic and molecular characterization of the enzyme.
6-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 two-phase 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.
Institut für Biochemie, Medizinische Fakultät der Universität Leipzig, Liebigstrasse 16, D-04103 Leipzig, Germany. kirj@medizin.uni-leipzig.de
Jürgen
J
Kirchberger
Jörg
J
Bär
Wolfgang
W
Schellenberger
Hassan
H
Dihazi
Gerhard
G
Kopperschläger
D000595:Amino Acid Sequence; D003470:Culture Media; D004789:Enzyme Activation; D005947:Glucose / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010732:Phosphofructokinase-1 / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D010843:Pichia / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development
D003470:Culture Media; D010732:Phosphofructokinase-1; D005947:Glucose