Abstract
Dihydroxyacetone kinase (DHAK) from the cell‐free extract of methanol‐grown Candida methylica was partially purified about 100‐fold by a procedure employing streptomycine sulfate fractionation, ammonium sulfate fractionation, negative absorption on Cibacron blue F3G‐A sephadex G 200 and DEAE‐cellulose column chromatography. The enzyme was stable in 50 mm Tris‐HCl buffer pH 7.5 containing 60% glycerol at –18°C.
The pH optimum for the activity of DHAK from C. methylica was 7.5. The purified enzyme phosphorylated dihydroxyacetone four times faster than d,l‐glyceraldehyde. The apparent Michaelis‐Menten constants for dihydroxyacetone and d,l‐glyceraldehyde were 0.011 mm and 0.024 mm. Other C~3~ compounds including glycerol were not phosphorylated. ITP and UTP were used as phosphate donors with a reaction rate of 11% and 3.1%, respectively, in relation to ATP, whereas the reaction rates of DHAK from C. methylica with CTP or GTP were much lower than 1%. The reaction of DHAK depends upon the presence of divalent cations in the assay. The highest activity was found with Mg^2+^ ions. The reaction rates with Co^2+^ or Ca^2+^ ions were only 57.3% and 30.3%, respectively, in relation to the assay with magnesium ions. Manganese chloride in the assay led to a complete loss of activity.