Dynamics of Substrate Processing by PPIP5K2, a Versatile Catalytic Machine.

Processing of substrates by enzymes can only be fully understood through their conformational dynamics; this is particularly true for the diphosphoinositol pentakisphosphate kinase PPIP5K2, an enzyme with critical roles in cell signaling and bioenergetic homeostasis. PPIP5K2 is remarkable for the reversible nature of its kinase activity, its unique ligand-stimulated ATPase activity, and the substrate traveling between two ligand-binding sites. Here we use molecular dynamics and data analysis techniques to rationalize these PPIP5K2 activities, thereby increasing our understanding of complex enzymatic mechanisms. In particular, we demonstrate how the enzyme's distinctive, ratchet-like mechanism harnesses the energy of random fluctuations to significantly reduce the entropy toll for intramolecular substrate transfer. We show that pre-reaction pulling forces along the reaction coordinate are predictive of the various PPIP5K2 catalytic activities. An unexpected possibility, raised by these computational studies, that 3,5-IP8 might be a substrate for dephosphorylation was experimentally interrogated and confirmed in a luciferase assay. • Molecular dynamics was used to rationalize the catalytic versatility of PPIP5K2 • A distinctive ratchet mechanism is involved in intramolecular substrate transfer • Enzyme-substrate forces are predictive of the various PPIP5K2 catalytic activities • A new substrate was computationally predicted and experimentally confirmed Remarkable catalytic versatility of the pyrophosphate kinase PPIP5K2 can only be fully understood through its conformational plasticity. An et al. used molecular dynamics to study its enzymatic mechanisms. They revealed a distinctive ratchet-like mechanism of substrate transfer and showed how enzyme-substrate forces explain the enzyme's catalytic activities. [ABSTRACT FROM AUTHOR]