ISSN:
1432-1017
Keywords:
Hexokinase (wheat germ)
;
conformational change, hysteresis
;
pulse fluorimetry
;
synchrotron radiation
Source:
Springer Online Journal Archives 1860-2000
Topics:
Biology
,
Physics
Notes:
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.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/BF00254211
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