TOPOGRAPHY RESEARCH OF THIAMINE KINASE ACTIVE CENTER IN PIG BRAIN USING FLUORESCENT PROBES

Abstract

Background. Biosynthesis of thiamine-diphosphate – coenzyme form of B 1 – is carried out with thiamine kinase (EC 2.7.6.2). The mechanism and ways of its regulation can be essentially clarified after identification of structural organization of enzyme active center and its conformational mobility. Objective. To study topography of thiamine kinase active center in pig brain and local conformation phenomena, which are responsible for protein functionality. Material and methods. Research has been carried out on electrophoretically homogeneous preparations of pig brain enzyme using 2-toluidinenaphthalene-6-sulfonate and 1-anilinonaphthalene-8-sulfonate as probes. Results. It has been established that active center of thiamine kinase is a hydrophobic cavity, where thiamine and ATP (adenosine triphosphate) are sorbed by pyrimidine and adenine cycles, and diphosphate radical of substrate- donor is directed to substrate-acceptor side. Pyruvate is connected where thiamine is located and facilitates elimination of thiamine diphosphate. Metal ions define optimal sorption geometry of substrates and, obviously, contribute to its convergence. Organization model of thiamine kinase catalytic center has been proposed. Conclusions. The sorption area of substrates, cofactors and effectors in enzyme molecule are spastically divided. Their interaction is implemented with conformational reorganization of globule.

Keywords

• t hiamine kinase
• pig brain
• fluorescent analysis
• thiamine kinase