Partial purification and properties of thiaminokinase from yeast

Abstract

Thiaminokinase, the enzyme catalyzing the synthesis of thiaminepyrophosphate from thiamine and adenosinetriphosphate, has been extracted from fresh bakers' yeast by plasmolysis by freezing at -70°C and thawing, followed by maceration at 37° in 0.5 M KCl.

The enzyme has been partially purified by fractionation of the yeast extract with ammonium sulphate. The active protein fraction has been subjected to fractional analysis according to . It is pointed out that the protein leaving the solution in the same range of salt concentration as does the enzymic activity is not homogeneous and probably does not represent the enzyme protein, but inert proteins which have adsorbed the enzyme - which will only constitute a small fraction of the precipitating protein - to the same extent.

Thiaminokinase requires Mg for its action. Optimal activation is obtained with concentrations of 2·10-2 M. Low concentrations of Mn also activate the enzyme, even more so than comparable amounts of Mg; but concentrations of Mn above 2·10-3 M markedly inhibit the phosphorylation of thiamine.

The action of the enzyme is stimulated strongly by small amounts of inorganic phosphate. Maximal effect is obtained with a concentration of 2·10-3 M.

The crude yeast extract contains at least two factors inhibiting thiaminokinase, which can be removed by dialysis, and are thermostable. One of these is of inorganic nature.

The enzyme deploys its maximal activity in the pH range of 6 to 8.

The purified preparation synthesizes thiaminepyrophosphate at a higher rate from thiamine than from thiamine monophosphate.