br Results br Discussion Members of the PfkB

Results
Discussion Members of the PfkB family of proteins, which exhibit AK activity, have only been found in the eukaryotic organisms in the past. Hence, the report of a PfkB family protein in MTub that exhibited AK activity was of much interest, and it necessitated closer examination of its characteristics. This MTub protein exhibited much higher sequence similarity to RKs and fructokinases than to AK from various sources. Further, the functional form of this MTub protein is a dimer [7] similar to RKs from various sources [16], [17], whereas all known AKs are monomers [18], [19]. Another aspect in which MTub-AK resembles RK more than other AKs is its ionic requirement. Like other RKs, the catalytic activity of MTub-AK is stimulated in the presence of monovalent metal ions such as K+[20], while AKs from various sources generally do not require monovalent ions for their activity [3]. On the other hand, the biological activity of various AK shows a strong dependence upon the presence of pentavalent ions [3], [11], which had no effect on the MTub protein. The results of various experiments presented here further illustrate that the MTub enzyme in question behaves very differently from other well-characterized AKs. Most importantly, this protein, when expressed either in bacterial or mammalian cells, failed to metabolize various Ado analogs to their toxic forms, while the AKs from human or CHO hcl name were very active in this regard. These results raise serious concerns whether this MTub protein indeed functions as AK in a cellular setting. In this context, it should be noted that although our results confirm that this MTub protein exhibits AK activity in vitro, we find this activity to be very low (about 320-fold lower) in comparison to human AK. This result disagrees with an earlier report by Long and Parker [21], who reported comparable specific activity of both human AK and the MTub enzymes for Ado. We believe that the catalytic activity of human AK in this earlier study was greatly underestimated in comparison to the MTub protein due to a number of reasons. The AK activity from various sources shows a marked dependence upon the presence of inorganic phosphate [11], which was not added to the enzyme reactions, whereas K+ required for the activation of the M. tuberculosis protein was [21]. Also, the assays for human AK were carried out at pH 6, while those for the M. tuberculosis protein were at pH 8. The M. tuberculosis protein functions optimally at pH 8 [7], whereas the human enzyme at pH 6 only shows half to a third of its maximal activity (pH 7.5–8.5) [22]. Another factor to consider in determining whether this MTub protein functions as AK in cells is the relative abundance of Ado and other simple sugars in cellular environment. Ado exists in micromolar amounts in cells [3], whereas sugars such as ribose and fructose are in millimolar ranges, as reflected in the Km values of the enzymes metabolizing cellular Ado and sugars [3]. Therefore, although the MTub protein in question shows 9-fold higher catalytic efficiency for Ado over ribose in vitro, which is unusual and it could be due to convergent evolution [7], it is more likely involved in the metabolism of ribose or some other sugar than Ado in a cellular environment where the sugar exists in great excess over Ado. In this context, the inferences from the recently reported crystal structure of the MTub protein [23] should also be interpreted carefully. Although this structure was solved with a bound Ado molecule in the active site, it is clear that ribose can also bind to the same site. Most of the residues (four out of five) that form polar contacts with the ribose moiety of Ado in MTub-AK (Asp12, Gly48, Asn52, and Asp257) are conserved in E. coli RK (Asp16, Gly42, Asn46, and Asp255) [18], [19], indicating that the same interaction will occur with ribose. Thus E. coli RK should also be able to bind Ado, since it shows ∼50-fold higher AK activity than MTub-AK. In contrast, the structure around the purine base of Ado in MTub-AK shows no similarity in the residue conservation and arrangement to other known structures of AK [18], [19].