THZ531 br Methods br Results and discussion br Conclusions A

Methods
Results and discussion
Conclusions A third example of a hexokinase using polyphosphate exclusively as a phosphoryl donor has been identified, this time in mammalian tissues. This is the first polyphosphate utilizing enzyme activity demonstrated in mammalian THZ531 as far as we are aware. The evolutionary significance of this enzyme may help in the understanding of the shift of substrate specificity of hexokinases from polyphosphate to ATP. The enzyme kinetics are similar to glucokinase as defined by its high S0.5 for glucose. Preliminary analysis suggests that the enzyme tightly binds polyphosphate which can act as a native substrate for glucose 6-phosphorylation. Because of the finite supply of polyphosphate in cells, the enzyme system is likely to be involved in acute cellular events. An obvious possibility, though speculative, is the supply of increased reductive capacity in the form of NADPH for glutathione synthesis during oxidative stress, supported by the observation that G6PDH localizes to the nuclear periphery [26].
Author contribution
Author information The authors declare no competing interests. Information requests, reprints and questions should be addressed to the corresponding author, S.B. ([email protected]).
Notes
Acknowledgements We thank The Wellcome Trust for funding to SPB (Post Doctoral funding for HB), and The Wellcome Trust for funding to D. Claire Wathes. We thank the BBSRC (01/A1/S/07269) for Ph.D. studentship funding for Angelina Swali, awarded to D. Claire Wathes. We thank Robert D. Cohen for valuable discussion concerning acid-base metabolism and regulation of gluconeogenesis, and Richard A. Iles for discussions on reversible reactions.
Introduction The ADP-dependent sugars kinases catalyze the transfer of a phosphoryl group from a di-phosphorylated nucleotide (ADP) to either glucose (EC:2.7.1.147) or fructose-6P (EC: 2.7.1.146). These enzymes belong to the ribokinase superfamily and share a common Rossmann-like fold that constitutes the named large domain. They also present an extra small domain, which has been proposed as a phylogenetic marker for the evolution of this superfamily. The ADP-dependent sugar kinases were initially described in archaea, but its presence in higher eukaryotes and some bacteria has been also demonstrated [1], [2], [3]. Regarding the catalytic mechanism of the ADP-dependent kinases, two highly conserved motifs have been described; the GXGD motif, which contains an aspartic acid residue that has been pointed out as the catalytic base that removes the proton from the acceptor hydroxyl group thus activating it for the nucleophilic attack [4], [5], [6], [7], [8] and the NXXE motif, involved in coordinating the metal cation, which recently has been related to the regulation of the enzyme activity [9]. Lately, a new motif has been reported for the ADP-dependent sugar kinases from archaea, the HXE motif. This motif has been described as fundamental for glucose binding during the formation of the ternary complex [9] and has not been reported for other members of the ribokinase family. So far, the best characterized are the thermophilic enzymes from the order Thermococcales, being the glucokinase (GK) and phosphofructokinase (PFK) enzymes highly specific for glucose [10] or fructose-6P, respectively [11]. Interestingly, in 2002, Sakuraba et al. [12] reported the characterization of the bifunctional enzyme from the hyperthermophilic methanogenic archeon Methanocaldococcus jannaschii.. In order to address if bi-functionality was a particular feature of the M. jannaschii enzyme or a common feature for all the enzymes from the order Methanococcales, Castro-Fernandez et al. [13] characterized the enzyme from Methanococcus maripaludis and constructed molecular models for all the ADP-dependent sugar kinases from the order Methanococcales. They concluded that all the enzymes from this order are able to phosphorylate glucose and fructose-6P, statement that is support by the fact that all the organisms from this order have only one copy of the gene codifying for the ADP-dependent enzymes.