adenosine-kinase and arsenic-acid

Ribokinase and adenosine kinase are both members of the PfkB family of carbohydrate kinases. The activity of mammalian adenosine kinase was previously shown to be affected by pentavalent ions (PVI). We now present evidence that the catalytic activity of E. coli ribokinase is also affected by PVI, increasing both the velocity and affinity of the enzyme for D-ribose. The Km, for ribose decreased from 0.61 mM to 0.21, 0.25, and 0.33 mM in the presence of 20 mM phosphate, arsenate, and vanadate, respectively. The activity of ribokinase was stimulated in a hyperbolic fashion, with the maximum velocity increasing 23-fold, 13-fold, and 11-fold under the same conditions, respectively. Activity was also affected upon the addition of phosphoenolpyruvate, suggesting that phosphorylated metabolites could be involved in enzymatic control. The similar effect of PVI on distantly related enzymes suggests that a common mechanism for activity is shared among PfkB family members.

Topics: Adenosine Kinase; Adenosine Triphosphate; Arsenates; Escherichia coli; Hydrogen-Ion Concentration; Ions; Phosphates; Phosphoenolpyruvate; Phosphotransferases (Alcohol Group Acceptor); Recombinant Proteins; Substrate Specificity; Vanadates

Purified adenosine kinase (AK) from Syrian hamster and bovine liver was examined for the presence of adenosine (Ad)-AMP exchange activity. The enzyme from both sources, in addition to catalyzing the conventional ATP-dependent phosphorylation of adenosine, supported an Ad-AMP exchange reaction that required ADP. Under optimal conditions both these reactions were found to occur at comparable rates. Several observations strongly indicate that the Ad-AMP exchange activity is an integral part of AK and it is likely associated with its catalytic mechanism. These observations include: (i) Both AK and Ad-AMP exchange activities show a nearly complete dependence upon the presence of pentavalent ions such as phosphate, arsenate or vanadate for catalysis; (ii) Both activities show similar heat-lability and inhibition by 5-iodotubercidin (5-ITu); (iii) In a Chinese hamster cell mutant resistant to adenosine analogs that lacked AK activity, the Ad-AMP activity was also found to be absent. The presence of a phosphoryl-enzyme intermediate, or any exchange between free 32Pi and any of the reactants, however, was not detected under the reaction conditions. Some implications of these observations regarding the catalytic mechanism of AK are discussed.

Topics: Adenosine; Adenosine Kinase; Adenosine Monophosphate; Adenosine Triphosphate; Animals; Arsenates; Cattle; CHO Cells; Cricetinae; Enzyme Inhibitors; Enzyme Stability; Kinetics; Liver; Magnesium; Mutation; Phosphates; Temperature; Toyocamycin; Tubercidin

The enzyme adenosine kinase (AK) has been purified to homogeneity from Syrian hamster and bovine livers. The purified enzymes from both these sources have a Mr of approximately 38 kDa, as determined by gel-filtration and SDS-polyacrylamide gel electrophoresis. A novel characteristic of AK observed here is that its catalytic activity shows a nearly complete dependence upon the presence of pentavalent ions such as phosphate (P(i)), arsenate or vanadate. Maximal AK activity was observed in the presence of either 2-3 mM P(i), or 5-10 mM arsenate, or 10-20 mM vanadate. A low basal level of AK activity (1-5% of maximal) observed in the absence of these ions is attributed to P(i) contamination in the adenine nucleotides preparations. The presence of P(i) had no effect on the K m for ATP (0.4 mM), but it markedly increased the affinity of the enzyme for adenosine. The K(m) of AK for adenosine in presence of 0, 0.1 mM and 2 mM P(i) was estimated to be 1.4 mu M, 0.77 mu M and 0.095 mu M, respectively. Free P(i) showed no exchange with any of the reactants during the assay conditions, and its presence had no effect on the thermostability of the enzyme. These observations suggest that the pentavalent ions such as phosphate may be playing an important role in the enzyme's catalytic mechanism by facilitating either binding of adenosine to the enzyme or in the formation of an enzyme-ATP-adenosine complex.

Topics: Adenosine Kinase; Animals; Arsenates; Cattle; Cricetinae; Kinetics; Liver; Mesocricetus; Vanadates