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PECULIARITIES OF FLUORESCENCE OF THIAMINE KINASE COVALENTLY LABELED WITH PYRIDOXAL-5-PHOSPHATE IN PIG BRAIN

Journal: Journal of the Grodno State Medical University (Vol.53, No. 1)

Publication Date:

Authors : ; ; ;

Page : 67-72

Keywords : thiamine kinase; pig brain; fluorescence analysis;

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Abstract

The properties and parameters of pig brain thiamine kinase (EC 2.7.6.2) during its interaction with substrates (thiamine, ATP), cofactor (magnesium ions) and effector (pyruvate) have been studied with the use of a fluorescent label, (pyridoxal 5’-phosphate). It has been shown that enzyme molecule contains two rapidly reacting centers binding the mark by ε- or N-terminals of lysine α-amino residues with Ks of 0,9?103 M-I. Modification of amino groups is accompanied by marked increase in enzymatic reaction rate, the rate variation indicating ambiguity of both sites for modification. Thiamine kinase activation with labeling may be explained by the presence of carbonyl group in it and is similar to pyruvate labeling activation by the mechanism. Pyruvate and enzyme substrate ?thiamine effectively inhibit the fluorescence of both reacting centers of chromophore labels regardless of their entry sequence which indicates functional relationship between sorption sites, realized through protein molecule conformation changes. Sorption sites for ATP and magnesium ions are spatially distant from the label.

Last modified: 2016-05-03 17:05:52