Characterization of Hexokinase and Fructokinase from Suspension-Cultured Catharanthus roseus Cells*

from Suspension-Cultured Catharanthus roseus Cells* Yüko Yamashita and Hiroshi Ashihara Department of Biology, Faculty of Science, Ochanomizu University, 2-1-1, Otsuka, Bunkyo-ku, Tokyo, 112, Japan Z. Naturforsch. 43c, 827-834 (1988); received June 28, 1988 Catharanthus roseus, Suspension Culture, Fructokinase, Glycolysis, Hexokinase Two different hexose-phosphorylating enzymes, hexokinase and fructokinase, were partially purified from suspension-cultured Catharanthus roseus cells. One of the enzymes, hexokinase, catalyzed the phosphorylation of both glucose and fructose. The Km values for glucose and fructose were 0.06 mM and 0.23 mM, respectively. The Vmax of the enzyme with fructose was approximately three times higher than with glucose. This enzyme was specific in its requirement for ATP and its Km value for ATP was 52 (ÍM. The optimum pH was 8.0 and Mg :+ or Mn : + was required for the activity. The activity was inhibited by considerably higher concentrations of A D P (i.e., 4 mM A D P was required for 50% inhibition). The second enzyme, fructokinase, was specific for fructose, and no activity was detected with glucose as substrate. This enzyme used UTP or CTP as phosphate donor. The Km values of this enzyme for fructose and UTP were 0.13 mM and 0.15 mM, respectively. The pH optimum was 7.2, and Mg or Mn was required for the activity. These divalent cations could be partially replaced by Ca. The activity was inhibited noncompetitively by A D P and AMP. 90% inhibition of the activity by 0.5 mM ADP was observed in the presence of 2 mM UTP and 5 mM MgCl2. Fructose-2,6-bisphosphate, glucose-1,6-bisphosphate, glucose-6-phosphate, and fructose-6-phosphate had little or no effect on the activity of both the hexokinase and the fructokinase. Based on these results, a discussion is presented of the role of hexokinase and fructokinase and their involvement in the regulation of the metabolism of sugars in Catharanthus cells.