The kinetic properties of a human PPIP5K reveal that its kinase activities are protected against the consequences of a deteriorating cellular bioenergetic environment

• Published in: Bioscience reports Vol. 33; no. 2; p. e00022
• Main Authors: Weaver, Jeremy D, Wang, Huanchen, Shears, Stephen B
• Format: Journal Article
• Language: English
• Published: England Portland Press Ltd 05.02.2013; Portland Press, Biochemical Society
• Subjects: bis-diphosphoinositol tetrakisphosphate; Catalysis; cellular energy homoeostasis; diphosphoinositol pentakisphosphate; diphosphoinositol polyphosphate; Energy Metabolism; Humans; Inositol - chemistry; Inositol - metabolism; inositol 1,3,4-trisphosphate 5/6-kinase (ITPK1); Inositol Phosphates; inositol pyrophosphate; Kinetics; Metabolic Networks and Pathways; Original Paper; Phosphates - chemistry; Phosphates - metabolism; Phosphotransferases (Phosphate Group Acceptor) - chemistry; Phosphotransferases (Phosphate Group Acceptor) - genetics; Phosphotransferases (Phosphate Group Acceptor) - metabolism; Substrate Specificity
• ISSN: 0144-8463; 1573-4935
• DOI: 10.1042/BSR20120115

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.