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  • The enzyme plays an important role

    2023-09-27

    The enzyme plays an important role in the purine metabolism of this parasite. Like other parasitic protozoa, T. gondii is incapable of de novo purine biosynthesis and depends totally on preformed purines salvaged from the host K03861 for its purine requirements and survival (Perrotto et al., 1971; Schwartzman and Pfefferkorn, 1982 and Krug et al., 1989). Ado is the best precursor for the synthesis of purine nucleotides in T. gondii and the salvage of Ado is extraordinarily efficient. Incorporation of Ado into purine nucleotides in T. gondii exceeds that of any other purine precursor by at least 10-fold (Pfefferkorn and Pfefferkorn, 1978 and Krug et al., 1989). Ado is converted to AMP, a branch point from which all purine nucleotides are synthesized. Metabolic, biochemical, molecular, and fitness studies demonstrated that TgAK is the route of this efficient metabolism of Ado to AMP (Pfefferkorn and Pfefferkorn, 1978, Krug et al., 1989 and Chaudhary et al., 2004) By contrast, most mammalian cells preferentially deaminate Ado to inosine; whereas, the deaminating enzyme, Ado deaminase (EC 3.5.4.4), is missing from or has very little activity in T. gondii (Krug et al., 1989). Moreover, comparative enzymatic, genetic, and metabolic studies showed that TgAK is the only purine nucleoside kinase in T. gondii (Pfefferkorn and Pfefferkorn, 1978, Krug et al., 1989, Iltzsch et al., 1995, el Kouni et al., 1999 and Chaudhary et al., 2004). TgAK does not phosphorylate any of the natural purine nucleosides other than Ado (Krug et al., 1989; Iltzsch et al., 1995 and el Kouni et al., 1999). However, TgAK, unlike human Ado kinase, can phosphorylate various 6-substituted-9-β-D-ribofuranosylpurine analogues to their respective nucleoside 5′-monophosphates (Iltzsch et al., 1995; Darling et al., 1999; el Kouni et al., 1999; Al Safarjalani et al., 2003, 2008, 2010; el Kouni, 2003, 2007; Yadav et al., 2004; Rais et al., 2005 and Kim et al., 2008, 2010). When certain 6-substituted-9-β-d-ribofuranosylpurine analogues are used as subversive substrates they are selectively phosphorylated by TgAK and become toxic only against the parasites but not their host (el Kouni et al., 1999; el Kouni, 2003, 2007; Yadav et al., 2004; Rais et al., 2005 and Al Safarjalani et al., 2008, 2010). It follows that TgAK represents an excellent target for the development of chemotherapeutic regimens for the treatment of toxoplasmosis with 6-substituted subversive substrates (el Kouni, 2003, 2007). Yet, in order to fully understand the remarkable efficiency of TgAK in the synthesis nucleotides of several 6-substituted-9-β-d-ribofuranosylpurine analogues as well as Ado, complete kinetic parameters of this enzyme would be necessary. Detailed kinetic studies are essential to fully understand the basic reaction mechanism of enzymes (e.g. ping-pong, sequential, random, ordered, etc.) and to illustrate the order of binding of the substrates and release of the products. The order of addition of substrates and the mechanism of action of the enzyme will shed some light on the topology of the active center. Detailed kinetic studies can also demonstrate whether either of the substrates displays substrate inhibition and/or non-linear product inhibition which would indicate whether there is more than one binding site on the enzyme for the binding of substrate(s). Such studies are also required to determine whether there is a “cooperative effect” between the substrates or not, which would aid in illustrating whether the enzyme has multiple interacting allosteric and/or catalytic sites. Furthermore, comparison with the kinetics of human Ado kinase would reveal a great deal about the structure and function of TgAK and will also aid in understanding the differences in substrate specificities between the two enzymes. Such information cannot be visualized by X-ray crystallography of the purified enzyme but could be critical in explaining the X-ray crystallographic results and the future design of subversive substrates. Therefore, in order to fully understand the role of TgAK in the metabolism Ado and 6-substituted purines in particular, as well as for future drug design, a complete kinetic study of this enzyme is performed.