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Polarised pulse fluorimetry study on the conformational properties of wheat germ hexokinase LI

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Abstract

The conformational properties of wheat germ hexokinase LI, a monomeric enzyme showing non-Michaelian kinetics, have been studied by polarised pulse fluorimetry using synchrotron radiation as an excitation light source.

The fluorescence decays and the fluorescence anisotropy decays of tryptophyl residues were measured with excitation at 300 nm. At pH 8.5, we found that the “mnemonical” temperature-dependent transition did not induce any detectable structural change in the protein. This rules out modifications of the aggregation state of hexokinase during the transition as well as important conformational changes in the tertiary structure. At pH 6.1, a temperature-dependent transition of the enzyme-glucose binary complex is observed: rapid, large amplitude, internal motions appear in the structure when the temperature is raised from-1°C to 30°C. Full standard activity is retained during this dynamic change.

In the experiments described here we obtained an active fluorescent derivative by reacting hexokinase with N-(iodoacetylaminoethyl)-5-naphtylamine-1-sulfonic acid (1,5-IAEDANS), in the presence of glucose. Polarised fluorescence decay measurements indicate that the label is exposed to the solvent and very mobile, which makes it ineffective as a probe for the conformational properties of hexokinase.

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Abbreviations

1,5-IAEDANS:

N-(iodoacetylaminoethyl)-5-napthylamine-1-sulfonic acid

DTNB:

5,5′-dithiobis(2-nitrobenzoic acid)

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Authors and Affiliations

  1. Laboratoire pour l'Utilisation du Rayonnement Electromagnétique, Université Paris Sud, Bât 209 C, F-91405, Orsay Cedex, France

    F. Merola & J. C. Brochon

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  1. F. Merola
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  2. J. C. Brochon
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Merola, F., Brochon, J.C. Polarised pulse fluorimetry study on the conformational properties of wheat germ hexokinase LI. Eur Biophys J 13, 291–299 (1986). https://doi.org/10.1007/BF00254211

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  • DOI: https://doi.org/10.1007/BF00254211

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