title: The kinetic properties of a human PPIP5K reveal that its kinase activities are protected against the consequences of a deteriorating cellular bioenergetic environment.
abstract: We obtained detailed kinetic characteristics--stoichiometry, reaction rates, substrate affinities and equilibrium conditions--of human PPIP5K2 (diphosphoinositol pentakisphosphate kinase 2). This enzyme synthesizes 'high-energy' PP-InsPs (diphosphoinositol polyphosphates) by metabolizing InsP₆ (inositol hexakisphosphate) and 5-InsP₇ (5-diphosphoinositol 1,2,3,4,6-pentakisphosphate) to 1-InsP₇ (1-diphosphoinositol 2,3,4,5,6-pentakisphosphate) and InsP₈ (1,5-bis-diphosphoinositol 2,3,4,6-tetrakisphosphate), respectively. These data increase our insight into the PPIP5K2 reaction mechanism and clarify the interface between PPIP5K catalytic activities and cellular bioenergetic status. For example, stochiometric analysis uncovered non-productive, substrate-stimulated ATPase activity (thus, approximately 2 and 1.2 ATP molecules are utilized to synthesize each molecule of 1-InsP₇ and InsP₈, respectively). Impaired ATPase activity of a PPIP5K2-K248A mutant increased atomic-level insight into the enzyme's reaction mechanism. We found PPIP5K2 to be fully reversible as an ATP-synthase in vitro, but our new data contradict previous perceptions that significant 'reversibility' occurs in vivo. PPIP5K2 was insensitive to physiological changes in either [AMP] or [ATP]/[ADP] ratios. Those data, together with adenine nucleotide kinetics (ATP Km=20-40 μM), reveal how insulated PPIP5K2 is from cellular bioenergetic challenges. Finally, the specificity constants for PPIP5K2 revise upwards by one-to-two orders of magnitude the inherent catalytic activities of this enzyme, and we show its equilibrium point favours 80-90% depletion of InsP₆/₅-InsP₇.
authors.affiliation: Inositol Signaling Group, Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, NIH, DHHS, Research Triangle Park, P.O. Box 12233, NC 27709, U.S.A.
authors.forename: Jeremy D
authors.initials: JD
authors.lastname: Weaver
authors.affiliation: Inositol Signaling Group, Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, NIH, DHHS, Research Triangle Park, P.O. Box 12233, NC 27709, U.S.A.
authors.forename: Huanchen
authors.initials: H
authors.lastname: Wang
authors.affiliation: Inositol Signaling Group, Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, NIH, DHHS, Research Triangle Park, P.O. Box 12233, NC 27709, U.S.A.
authors.forename: Stephen B
authors.initials: SB
authors.lastname: Shears
mesh_terms: D002384:Catalysis; D004734:Energy Metabolism; D006801:Humans; D007294:Inositol / Q000737:chemistry / Q000378:metabolism; D007295:Inositol Phosphates; D007700:Kinetics; D053858:Metabolic Networks and Pathways; D010710:Phosphates / Q000737:chemistry / Q000378:metabolism; D017856:Phosphotransferases (Phosphate Group Acceptor) / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D007295:Inositol Phosphates; D010710:Phosphates; C402123:inositol heptakisphosphate; D007294:Inositol; D017856:Phosphotransferases (Phosphate Group Acceptor); C562140:PPIP5K2 protein, human