Subunit dissociation and inactivation of pyruvate kinase by hydrostatic pressure oxidation of sulfhydryl groups and ligand effects on enzyme stability.
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+, Mg2+ 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.
De Felice
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FG
Departamento de Bioquímica Médica, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Brazil.
Soares
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VC
Ferreira
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ST
D000818:Animals; D002942:Circular Dichroism; D019281:Dimerization; D004229:Dithiothreitol / Q000494:pharmacology; D006874:Hydrostatic Pressure; D007527:Isoenzymes / Q000737:chemistry* / Q000187:drug effects; D008024:Ligands; D009124:Muscle Proteins / Q000737:chemistry* / Q000187:drug effects; D010084:Oxidation-Reduction; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D011770:Pyruvate Kinase / Q000737:chemistry* / Q000187:drug effects; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013438:Sulfhydryl Compounds / Q000378:metabolism
D007527:Isoenzymes; D008024:Ligands; D009124:Muscle Proteins; D013438:Sulfhydryl Compounds; D011770:Pyruvate Kinase; D004229:Dithiothreitol