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Metabolism of D-Arabinose: Origin of a D-Ribulokinase Activity in Escherichia coli¹

^a Department of Microbiology, University of Massachusetts, Amherst, Massachusetts, 01002

ABSTRACT

The kinase responsible for the phosphorylation of D-ribulose was purified 45.5-fold from a strain of Escherichia coli K-12 capable of growth on D-arabinose with no separation of D-ribulo- or L-fuculokinase activities. Throughout the purification, the ratios of activities remained essentially constant. A nonadditive effect of combining both substrates in an assay mixture; identical K_m values for adenosine triphosphate with either L-fuculose or D-ribulose as substrate; and, the irreversible loss of activity on both substrates, after removal of magnesium ions from the enzyme preparation, suggest that the dual activity is due to the same enzyme. A fourfold greater affinity of the enzyme for L-fuculose than for D-ribulose, as well as a higher relative activity on L-fuculose, suggest that the natural substrate for this enzyme is L-fuculose. The product of the purified enzyme, with D-ribulose as substrate, was prepared. The ratio of total phosphorous to ribulose phosphate was 1.01:1, indicating that the product was ribulose monophosphate. The behavior of the kinase product in the cysteine-carbazole and orcinol reactions, as well as the results of periodate oxidation assays, provided evidence that it was not D-ribulose-5-phosphate. Reaction of this compound with a cell-free extract of E. coli possessing L-fuculose-l-phosphate aldolase activity resulted in the production of dihydroxyacetone phosphate and glycolaldehyde. The kinase product failed to reduce 2,3,5-triphenyltetrazolium and possessed a half-life of approximately 1.5 min in the presence of 1 N HCl at 100 C. These properties suggested that the phosphate group was attached to carbon atom 1 of D-ribulose.

² Present address: Department of Microbiology, Georgetown University Medical School, Washington, D.C. 20007.

¹ This paper was presented in part at the 70th Annual Meeting of the American Society for Microbiology, Boston, Mass. 26 April-1 May 1970.