Partial purification and properties of thiaminokinase from yeast.

It has been known for some time that living yeast is able to take up large amounts of thiamine, thereby converting this compound into thiaminepyrophosphate (TPP) I, 3, 3, 4,5. Normally, most of the thiamine is found to be present as T P P in yeast6; part of the latter compound can be accounted for as carboxylase. Very little additional carboxylase is formed when fresh yeast is incubated with thiamine~; most of the accumulated T P P is bound in some other manner 5. A few years ago we have studied the synthesis of TP P from thiamine by living yeast 5 and have realized that it is a complicated process. We have thought to gain more insight by first studying a more simple system, namely the phosphorylation of thiamine by the isolated enzyme in cell-free extracts or purified preparations. This field has already been explored by several workers. WEIL-MALHERBE s prepared a protein fraction from Lebedew juice of dried brewers' yeast which could transform thiamine into thiaminepyrophosphate and was the first to show that this phosphorylation required adenosinetriphosphate (ATP) as phosphate donator. His work was confirmed and extended by NGUYEN VAI~ THOAI AND CI-IEVILLARD 9, who prepared the enzyme phosphorylating thiamine from brewers' yeast and also from dog liver. Recent investigations by NIELSEN AND LEUTHARDT x° should also be mentioned although they do not concern the yeast enzyme; these authors used preparations from rat liver and showed that the phosphorylating enzyme is a soluble protein. We have extracted the enzyme from fresh bakers' yeast and have studied the phosphorylating activity of these yeast extracts and of partially purified protein fractions. The enzyme proved to require Mg++ ions. Small amounts of inorganic phosphate, though not indispensable, greatly enhanced the enzymic activity. As regards the influence of the pH, a broad optimum was found between pH 6 and 8. Further purification of the enzyme is in progress, but we have deemed it useful to set down here the most important observations so far obtained. In keeping with LIPMANN'S nomenclature u, we propose to give this enzyme, which transfers phosphate from ATP to thiamine, the name of thiaminokinase.