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Issue 15, 1993
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Biophysical study of halophilic malate dehydrogenase in solution: revised subunit structure and solvent interactions of native and recombinant enzyme

Abstract

In previous work, malate dehydrogenase from Haloarcula marismortui(hMDH), a halophilic enzyme that is only stable in high concentrations of certain salts, was characterized by different biophysical methods as a dimer of molar mass 87 000 g mol–1 with solvent interactions in multimolar NaCl solutions that are significantly larger than for non-halophilic proteins. A model was proposed in which hMDH is stabilized by different mechanisms in different salt solvents (cf. Eisenberg et al., Adv. Protein Chem., 1992, 43, 1). Recently, the gene coding for hMDH was isolated and sequenced and the recombinant protein expressed in E. coli and renatured (Cendrin et al., Biochemistry, 1993, submitted). A subunit molar mass of 32 638 g mol–1 was calculated from the sequence and confirmed by mass spectrometry. The present study was undertaken in order to resolve the discrepancy between this value and the previously proposed dimer solution structure. The absorption coefficient of the protein was redetermined by amino acid analysis. New densimetry measurements in a large range of salt concentrations, ultracentrifugation and light scattering experiments were performed on the native and recombinant enzymes, and previous ultracentrifugation, X-ray and neutron scattering data were re-analysed. Control experiments on bovine serum albumin (as a model for non-halophilic proteins) were also repeated under similar conditions. All the data are now in agreement with a tetrametric solution structure for hMDH, with solvent interactions (e.g. in multimolar NaCl or KCl solutions: ca. 0.4 g water and 0.1 g salt per g protein) that show water binding comparable to non-halophilic proteins yet an order of magnitude more salt binding than for non-halophilic proteins. The stabilization model for hMDH remains valid. The complementarity and accuracy of the different methods for solution structure analysis are discussed critically in the light of this study.

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Article type: Paper
DOI: 10.1039/FT9938902659
J. Chem. Soc., Faraday Trans., 1993,89, 2659-2666

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    Biophysical study of halophilic malate dehydrogenase in solution: revised subunit structure and solvent interactions of native and recombinant enzyme

    F. Bonneté, C. Ebel, G. Zaccai and H. Eisenberg, J. Chem. Soc., Faraday Trans., 1993, 89, 2659
    DOI: 10.1039/FT9938902659

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