Subunit dissociation and inactivation of pyruvate kinase by hydrostatic pressure

Abstract

The effect of hydrostatic pressure on the stability of tetrameric rabbit muscle pyruvate kinase was investigated by enzyme activity measurements, size-exclusion chromatography, circular dichroism and fluorescence spectroscopies. Under nonreducing conditions, enzyme activity was irreversibly inhibited by increasing pressure and was completely abolished at 350 MPa. Inhibition was dependent on the concentration of pyruvate kinase, indicating that it was related to pressure-induced subunit dissociation. Size-exclusion chromatography of pressurized samples confirmed a decrease in the proportion of tetramers and an increase in monomers relative to native samples. Addition of dithiothreitol immediately following pressure release led to full recovery of both enzyme activity and of native tetramers. Furthermore, no irreversible inhibition of pyruvate kinase was observed if pressure treatment was carried out in the presence of dithiothreitol. These data suggest that pressure-dissociated monomers undergo conformational changes leading to oxidation of sulfhydryl groups, which prevents correct refolding of native tetramers on decompression. These conformational changes are relatively subtle, as indicated by the lack of significant changes in far-UV circular dichroism and intrinsic fluorescence emission spectra of previously pressurized samples. The effects of various physiological ligands on the pressure stability of pyruvate kinase were also investigated. A slight protection against inhibition was observed in the simultaneous presence of K + , Mg 2+ and ADP. Both phosphoenolpyruvate and the allosteric inhibitor, phenylalanine, caused marked stabilization against pressure, suggesting significant energy coupling between binding of these ligands and stabilization of the tetramer.Keywords: pyruvate kinase; hydrostatic pressure; ligand binding; subunit dissociation; inactivation.Pyruvate kinase is an important regulatory glycolytic enzyme that catalyses the transfer of the phosphoryl group from phosphoenolpyruvate (PEP) to ADP, generating ATP and pyruvate. Pyruvate kinase requires both K and Mg 2 for activity [1±6]. In mammals, four different isozymes of pyruvate kinase that have different kinetic properties exist. The muscle isozyme (M1) is a homotetramer of 59 kDa subunits and does not exhibit cooperative behavior [7]. The kinetic mechanisms of pyruvate kinase from various sources and organisms have been investigated in detail [8±13]. The three-dimensional structure of rabbit muscle pyruvate kinase complexed with K + , Mn 2+ and pyruvate has recently been determined [14].The unfolding and refolding of pyruvate kinase by denaturing agents such as guanidine hydrochloride and urea have been investigated and were shown to be only partially reversible [15±20]. Hydrostatic pressure has been increasingly used to study protein subunit interactions and stability (reviewed in [21±23]). Protein oligomers ranging from dimers to large aggregates such as viral particles have been shown to undergo pressure-induced subunit dissociation. Press...

Selective-Targeted Effect of High-Pressure Processing on Proteins Related to Quality: a Proteomics Evidence in Atlantic Mackerel (Scomber scombrus)

“…However, the increase of b2 band seems to be due to a pressure-induced dissociation of tetramer pyruvate kinase, composed by 58 KDa pyruvate kinase monomers. In agreement with this, a previous investigation showed that HHP treatment causes dissociation and inactivation of pyruvate kinase (De Felice et al 1999). This effect of the HHP strength on sarcoplasmic proteins was not altered after a 3-month storage at -10 ºC.…”

A critical review of the role of M2PYK in the Warburg effect

“…The observation of the stable dimer is relevant to our current consideration [171][172][173][174], given the focus on the tetramer to dimer transition in studies of M 2 PYK in cancer. However, indications that the dimeric form of M 1 PYK is active are thought to be artifacts because PEP stabilizes the tetrameric form of that enzyme [173,175,176]. Therefore, even when isolated dimers of the M 1 PYK protein are added to an assay, the presence of PEP in the assay causes dimers to rapid assemble into active tetramers.…”

Large-Scale Transposon Mutagenesis ofPhotobacterium profundumSS9 Reveals New Genetic Loci Important for Growth at Low Temperature and High Pressure

“…Pyruvate kinase has previously been found to be a pressure-sensitive enzyme and to undergo adaptational changes in deep-sea animals (26,57). The requirement for this isozyme could have been a reflection of the medium conditions employed (growth under glucosefermenting conditions).…”