Dihydroxyacetone kinase of methanol-assimilating yeasts. II. Partial purification and some properties of dihydroxyacetone kinase from Candida methylica.
Dihydroxyacetone kinase (DHAK) from the cell-free extract of methanol-grown Candida methylica was partially purified about 100-fold by a procedure employing streptomycin 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 degrees 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 C3 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 Mg2+ ions. The reaction rates with Co2+ or Ca2+ 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.
D002175:Candida / Q000201:enzymology*; D002413:Cations, Divalent / Q000494:pharmacology; D004098:Dihydroxyacetone / Q000302:isolation & purification / Q000378:metabolism; D005985:Glyceraldehyde / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010770:Phosphotransferases / Q000302:isolation & purification / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor); D013379:Substrate Specificity
D002413:Cations, Divalent; D005985:Glyceraldehyde; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C023132:glycerone kinase; D004098:Dihydroxyacetone