Limited proteolysis of yeast phosphofructokinase

Abstract

Purified phosphofructokinase 1 from baker's yeast (Saccharomyces cerevisiae) was subjected to proteol ysis by thermolysin, endoproteinase lys-C, trypsin and chymotrypsin under defined solvent conditions. In the absence of substrates and allosteric effectors, the catalytic activity of phosphofructokinase rapidly disappeared in the presence of each proteolytic enzyme. The presence of a saturating concentration of ATP protected phosphofructokinase activity from proteolytic inactivation while the collective presence of fructose 6-phosphate, AMP and fructose 2,6-bisphosphate provided transient activation during proteolysis.Changes in the quaternary structure of phosphofructokinase resulting from proteolysis were estimated by high performance size exclusion chromatography while changes in the primary sequence of the individual a and p polypeptide chains were estimated by polyacrylamide-gel electrophoresis in sodium dodecylsulfate. The site(s) of proteolytic cleavage were identified by N-terminal sequence analysis of resolved electrophoretic components.The presence of ATP protects phosphofructokinase from thermolysin proteolysis, while the collective presence of fructose 6-phosphate, AMP and fructose 2,6-bisphosphate restricts proteolysis to one site in each polypeptide chain involving the peptide bonds preceding Leu199 in the a chain and Leu192 in the p chain. The truncated phosphofructokinase retains its octameric structure. The presence of ATP largely restricts endoproteinase lys-C proteolysis to a single site in the n chain involving the peptide bond preceding Va1914. This cleavage results in the dissociation of the octameric form of phosphofructokinase into two tetramers. The presence of ATP restricts both trypsin and chymotrypsin proteolysis to the N-terminal and C-terminal regions described above, resulting in the preferential stabilization of the tetrameric form of phosphofructokinase.It would appear that the first 200 and last 80 residues which are unique to the sequence of the yeast phosphosphofructokinase are not directly involved in catalysis or its allosteric regulation.However, the last 80 residues of the a polypeptide chain do appear to stabilize an octameric structure which is unique to yeast phosphofructokinase.Yeast phosphofructokinase 1 contains two kinds of polypeptide chains, an a chain containing 987 residues in a known sequence and encoded by the PFKl gene and a chain containing 959 residues, also in a known sequence encoded by the PFK2 gene (Heinisch et a]., 1989). The native enzyme is a globular protein containing four a chains and four p chains. Solution measurements indicate the native octameric enzyme has a sedimentation coefficient of 21 S and a molecular mass of about 800 kDa (Kopperschlager et al., 1977;Chaffotte et al., 1984). The octameric structure of the native enzyme is stable at a protein concentration of 1 mg/ ml at ambient temperature and neutral pH in the presence or Correspondence to

Single Point Mutations in Either Gene Encoding the Subunits of the Heterooctameric Yeast Phosphofructokinase Abolish Allosteric Inhibition by ATP

“…It has been observed that the presence of ATP protects Pfk in vitro from proteolytic inactivation (41). This has been proposed to be caused by conformational changes upon ATP binding.…”

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

“…The mobility was related to the molecular masses of the premixed protein high-range molecular weight marker from Roche Molecular Biochemicals and purified native (21 S) as well as proteolytically degraded Pfk (12 S), for which molecular masses are well known (Kopperschläger et al, 1993). 1D-Gel analysis was done with the scanned SDS-PAGE images by using Labimage software version 2.6 (Kapelan GmbH, Halle, Germany).…”

“…Denaturation of protein and SDS-PAGE were carried out according to Laemmli in 7.5% acrylamide running gels as described elsewhere (Kopperschläger et al, 1993) using the Mini-Protean II Dual Slab Cell system (Bio-Rad Laboratories). The mobility was related to the molecular masses of the premixed protein high-range molecular weight marker from Roche Molecular Biochemicals and purified native (21 S) as well as proteolytically degraded Pfk (12 S), for which molecular masses are well known (Kopperschläger et al, 1993).…”

A single point mutation leads to an instability of the hetero-octameric structure of yeast phosphofructokinase

“…The stability of the oligomeric structure has been studied by means of different methods [9,11,12]. In dilution, the native enzyme was found to be a stable octamer down to nanomolar protein concentrations in the presence of ammonium sulphate.…”

“…In contrast to the enzyme from most other organisms, which functions as homo-and heterotetramers, the yeast enzyme is a hetero-octamer composed of four α-and four β-subunits, which are arranged as a dimer of tetramers [6][7][8]. The subunits are each subject to limited proteolytic degradation in itro, involving primarily the N-terminal region of about 200 amino acids [9]. Careful preparation of phosphofructokinase in the presence of protease inhibitors, however, yields exclusively unprocessed subunits [10].…”