Pentavalent Ions Dependency Is a Conserved Property of Adenosine Kinase from Diverse Sources:  Identification of a Novel Motif Implicated in Phosphate and Magnesium Ion Binding and Substrate Inhibition

Abstract

The catalytic activity of adenosine kinase (AK) from mammalian sources has previously been shown to exhibit a marked dependency upon the presence of pentavalent ions (PVI), such as phosphate (PO4), arsenate, or vanadate. We now show that the activity of AK from diverse sources, including plant, yeast, and protist species, is also markedly enhanced in the presence of PVI. In all cases, PO4 or other PVI exerted their effects primarily by decreasing the Km for adenosine and alleviating the inhibition caused by high concentrations of substrates. These results provide evidence that PVI dependency is a conserved property of AK and perhaps of the PfkB family of carbohydrate kinases which includes AK. On the basis of sequence alignments, we have identified a conserved motif NXXE within the PfkB family. The N and E of this motif make close contacts with Mg2+ and PO4 ions in the crystal structures of AK and bacterial ribokinase (another PfkB member which shows PVI dependency), implicating these residues in their binding. Site-directed mutagenesis of these residues in Chinese hamster AK have resulted in active proteins with greatly altered phosphate stimulation and substrate inhibition characteristics. The N239Q mutation leads to the formation of an active protein whose activity was not stimulated by PO4 or inhibited by high concentrations of adenosine or ATP. The activity of the E242D mutant protein was also not significantly altered in the presence of phosphate. Although PO4 had no effect on the KmAdenosine for this mutant, the KmATP, K(i)Adenosine, and K(i)ATP were significantly decreased. In contrast to these mutations, N239L or E242L mutant proteins showed greatly decreased activity with an altered Mg2+ requirement. These observations support the view that N239 and E242 play an important role in the binding of PO4 and Mg2+ ions required for the catalytic activity of adenosine kinase.

Expression in Escherichia coli of a recombinant adenosine kinase from Saccharomyces cerevisiae: purification, kinetics and substrate analyses

“…This is similar to that found for the Toxoplasma gondii enzyme (2 µM) (Darling et al, 1999) and significantly higher than those measured from human (41 nM) (Spychala et al, Recombinant adenosine kinase from Saccharomyces cerevisiae 1149 1996) or plants (0.3-0.5 µM) AKs (Moffatt et al, 2000). In contrast, a ten-fold higher K m value for A (26 µM) was obtained when whole yeast extract was used as the enzyme source (Maj et al, 2002). These differences could be explained by the dissimilar reaction conditions, including ATP : Mg molar ratio, free ions and pH, that were employed with each yeast enzyme preparation.…”

“…An important feature of AK regulation is its inhibition by high adenosine concentrations. Its dependence on the ATP : Mg 2+ ratio and on the presence of inorganic phosphate and other ions has also been reported (Maj et al, 2000(Maj et al, , 2002. Concerning yeasts, a Saccharomyces cerevisiae AK was purified (Leibach et al, 1971).…”

Structure–activity relationship for adenosine kinase from Mycobacterium tuberculosis

“…Indeed, M. tuberculosis Ado kinase shared more sequence homology with ribokinases than other Ado kinases [10]. Most Ado kinases are monomeric proteins with a molecular mass between 35 and 40 kDa, and their activity is usually stimulated in the presence of inorganic phosphate (P i ) [15,18,19]. In contrast, Ado kinase from M. tuberculosis is a homodimer comprised of identical 35 kDa subunits [10,20,21].…”

Identification and characterization of human ribokinase and comparison of its properties with E. coli ribokinase and human adenosine kinase

“…which describes the rate of a bisubstrate reaction where the concentration of an inhibitory substrate A is varied at a fixed concentration of the other substrate [14,15]. For the K m determination of ribose, the concentration of ATP was held fixed at 5 mM, whereas the K m determination of ATP was carried out in the presence of 2 mM ribose.…”

“…Earlier studies on AK have identified several phosphorylated metabolites and related compounds, which can either activate AK in place of inorganic phosphate or inhibit the enzyme in the presence of phosphate [17,18]. Kinetic and structural studies indicate that these compounds and inorganic phosphate exert their effects by acting on the same regulatory site on the enzyme [14,17,18]. Therefore, it was of great interest to determine whether AK activators and inhibitors had similar effects on RK, which presumably shares a similar catalytic mechanism with AK.…”