On the purification and properties of hepatic ketohexokinase

Abstract

Several assays for the purifícation of liver ketohexokinase (ATP : D-fructose 1-phosphotransferase) are described.

A thermic treatment at 60° C is effective in separating much inert protein without sensible yield detriment if it is applied upon scantily purified preparations.

The enrichments got by positive adsorption upon calcium phosphate gel, as well as by fractionation with ethanol, are not casily reporducible. Digestión by papain is not aplicable, owing to destruction of the enzyme.

A method based on the Parks and col. technique is adopted, which gives, very quickly, preparations of notable activity, 35- 40 times higher than that of the initial homogenate. Very stable preparations are obtained by lyophilization.

The reaction follows the Michaelis-Menten kinetics well, and the Km valúes found are not substantially different from those previously described.

The presence in L-position of the —OH in C3 had been shown as a substrate requirement for ketohexokinase. Now it has been proved that introduction of a methyl in this group prevents phosphorylation of the sugar and the affinity for theenzyme is los.t. 3-methyl fructose is not phosphorylated, níƒor does it inhibit the phosphorylation of fructose.

The ketohexokinase has essential HS-groups, as has been shown vlith several specific reagents. Dinitrofluorobenzene also inhibits the enzyme, suggesting a possible role of the free H2N- groups in its action mechanism.