Phosphorylation of glycerol and dihydroxyacetone in Acetobacter xylinum and its possible regulatory role.
Extracts of Acetobacter xylinum catalyze the phosphorylation of glycerol and dihydroxyacetone (DHA) by adenosine 5'-triphosphate (ATP) to form, respectively, L-alpha-glycerophosphate and DHA phosphate. The ability to promote phosphorylation of glycerol and DHA was higher in glycerol-grown cells than in glucose- or succinate-grown cells. The activity of glycerol kinase in extracts is compatible with the overall rate of glycerol oxidation in vivo. The glycerol-DHA kinase has been purified 210-fold from extracts, and its molecular weight was determined to be 50,000 by gel filtration. The glycerol kinase to DHA kinase activity ratio remained essentially constant at 1.6 at all stages of purification. The optimal pH for both reactions was 8.4 to 9.2. Reaction rates with the purified enzyme were hyperbolic functions of glycerol, DHA, and ATP. The Km for glycerol is 0.5 mM and that for DHA is 5 mM; both are independent of the ATP concentration. The Km for ATP in both kinase reactions is 0.5 mM and is independent of glycerol and DHA concentrations. Glycerol and DHA are competitive substrates with Ki values equal to their respective Km values as substrates. D-Glyceraldehyde and l-Glyceraldehyde were not phosphorylated and did not inhibit the enzyme. Among the nucleotide triphosphates tested, only ATP was active as the phosphoryl group donor. Fructose diphosphate (FDP) inhibited both kinase activities competitively with respect to ATP (Ki= 0.02 mM) and noncompetitively with respect to glycerol and DHA. Adenosine 5'-diphosphate (ADP) and adenosine 5'-monophosphate (AMP) inhibited both enzymic activities competitively with respect to ATP (Ki (ADP) = 0.4 mM; Ki (AMP) =0.25 mM). A. xylinum cells with a high FDP content did not grow on glycerol. Depletion of cellular FDP by starvation enabled rapid growth on glycerol. It is concluded that a single enzyme from A. xylinum is responsible for the phosphorylation of both glycerol and DHA. This as well as the sensitivity of the enzyme to inhibition by FDP and AMP suggest that it has a regulatory role in glycerol metabolism.
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000249:Adenosine Monophosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism; D004098:Dihydroxyacetone / Q000378:metabolism*; D004099:Dihydroxyacetone Phosphate / Q000096:biosynthesis; D005636:Fructosephosphates / Q000494:pharmacology; D041822:Gluconacetobacter xylinus / Q000201:enzymology / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism; D005990:Glycerol / Q000378:metabolism*; D005991:Glycerol Kinase / Q000378:metabolism; D005994:Glycerophosphates / Q000096:biosynthesis; D006902:Hydroxymercuribenzoates / Q000494:pharmacology; D008274:Magnesium / Q000494:pharmacology; D008970:Molecular Weight; D010770:Phosphotransferases / Q000378:metabolism; D013386:Succinates / Q000378:metabolism; D014306:Trioses / Q000378:metabolism*
D005636:Fructosephosphates; D005994:Glycerophosphates; D006902:Hydroxymercuribenzoates; D013386:Succinates; D014306:Trioses; D000249:Adenosine Monophosphate; D004099:Dihydroxyacetone Phosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D010770:Phosphotransferases; D005991:Glycerol Kinase; D008274:Magnesium; D005947:Glucose; D004098:Dihydroxyacetone; D005990:Glycerol