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  • Biochemically the primary function of PPIP K

    2021-04-14

    Biochemically, Oxonic acid potassium salt receptor the primary function of PPIP5K2 is 1-kinase-mediated phosphorylation of 5-IP7 to form 1,5-IP8 (Shears, 2018). To a lesser extent, PPIP5K2 also phosphorylates IP6 to produce 1-IP7. Furthermore, PPIP5K2 has been shown to catalyze a reverse reaction, that is, dephosphorylation of 1,5-IP8 to form 5-IP7 through transfer of a phosphate group to Oxonic acid potassium salt receptor (Wang et al., 2012). Moreover, PPIP5K2 shows an incongruous substrate-stimulated ATPase activity. In addition, PPIP5K2 features a peculiar two-step mechanism of substrate processing. The first step requires PP-IP substrate to interact with a “capture site,” a basic patch on the protein surface that imposes fewer electrostatic and structural constraints on ligand binding than does the catalytic pocket (Wang et al., 2014). In the second step, the substrate is transferred ∼8 Å to the reaction site, to an orientation that is optimal to accept the γ-phosphate of ATP.
    Results
    Discussion In this study, we have used MD simulations to demonstrate how conformational dynamics rationalize previously intractable aspects of a singularly remarkable molecular machine: the PPIP5K kinase domain. This enzyme's catalytic activity is reversible, it exhibits a rare ligand-stimulated ATPase activity, and the reaction process requires higher polar substrate to traverse ∼8 Å between two ligand-binding sites (Wang et al., 2012, Wang et al., 2014). Our conclusions could not have readily been derived from structural data alone. In fact, the crystal structures used as starting points for our simulations were in general structurally far from the quasi-equilibrium system configurations. Here, we report on an attempt to quantify the microscopic pulling forces that are required for a phosphotransfer reaction. While the force analysis proved to be a useful qualitative tool to rationalize the enzymatic activity, we also sought to assess how realistic are the quantitative levels of forces calculated in this study. Experimental assessment of interatomic forces inside a protein is not yet technically possible. However, there are rupture forces relevant to our system that have been measured by atomic force microscopy (AFM): ∼180 pN for hydrogen bonds to ∼1,000 pN for nucleotide-nucleotide interactions to ∼2,000 pN for a covalent bond (Boland and Ratner, 1995, Grandbois et al., 1999, Williams et al., 1996). In this study, highest net forces, averaged over 50 ns, ranged between ∼1,000 pN in the reverse reaction system with the 1,5-IP8 substrate to ∼2,700 pN in the forward reaction system (the median values over all simulations, for the above systems, were respectively −1,026 pN and 2,200 pN). Thus, the forces observed in our simulations are consistent with those needed to either rupture a covalent bond (∼2,000 pN) or break an ionic interaction between basic amino acid side chains and the acidic co-factor (expected to be higher than a nucleotide-nucleotide interaction of ∼1,000 pN). Our data are also compatible with a computational study of residue-residue interactions inside the 3-phosphoglycerate kinase core, where instantaneous forces reached thousands of piconewtons, while forces averaged over all MD trajectories were on the order of hundreds of piconewtons (Palmai et al., 2014).
    STAR★Methods
    Acknowledgments This work was supported by the National Institutes of Health (grant 5R01DK101645-02), the UNC Eshelman Institute for Innovation (grant RX03712105), and the Intramural Research Program of the NIH/National Institute of Environmental Health Sciences.
    Introduction Based primarily on a highly conserved AMP-binding motif, computational investigations have identified a large number of putative CoA synthetases in the model plant Arabidopsis. The identification of such putative AAEs has allowed for the methodical testing of numerous compounds as potential substrates of the individual CoA synthetases. This approach has led to the determination of substrate specificities for a number of AAEs including AAE3, which has been determined to be an oxalyl-CoA synthetase shown to catalyze the first step in a previously uncharacterized pathway of oxalate catabolism [1]. Since this initial finding, recent studies have shown that yeast [2] and a number of plants including Medicago truncatula [3], rice bean [4], rice [5], buckwheat [6], and amaranth [6] express an oxalyl-CoA synthetase suggesting the fundamental importance of this enzyme to plants as well as other organisms.