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.

An Y ，Jessen HJ ，Wang H ，Shears SB ，Kireev D ... -  《-》

Synthetic inositol phosphate analogs reveal that PPIP5K2 has a surface-mounted substrate capture site that is a target for drug discovery.

Diphosphoinositol pentakisphosphate kinase 2 (PPIP5K2) is one of the mammalian PPIP5K isoforms responsible for synthesis of diphosphoinositol polyphosphates (inositol pyrophosphates; PP-InsPs), regulatory molecules that function at the interface of cell signaling and organismic homeostasis. The development of drugs that inhibit PPIP5K2 could have both experimental and therapeutic applications. Here, we describe a synthetic strategy for producing naturally occurring 5-PP-InsP4, as well as several inositol polyphosphate analogs, and we study their interactions with PPIP5K2 using biochemical and structural approaches. These experiments uncover an additional ligand-binding site on the surface of PPIP5K2, adjacent to the catalytic pocket. This site facilitates substrate capture from the bulk phase, prior to transfer into the catalytic pocket. In addition to demonstrating a "catch-and-pass" reaction mechanism in a small molecule kinase, we demonstrate that binding of our analogs to the substrate capture site inhibits PPIP5K2. This work suggests that the substrate-binding site offers new opportunities for targeted drug design.

Wang H ，Godage HY ，Riley AM ，Weaver JD ，Shears SB ，Potter BV ... -  《-》

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

Weaver JD ，Wang H ，Shears SB 《-》

First synthetic analogues of diphosphoinositol polyphosphates: interaction with PP-InsP5 kinase.

Riley AM ，Wang H ，Weaver JD ，Shears SB ，Potter BV ... -  《-》

Structural basis for an inositol pyrophosphate kinase surmounting phosphate crowding.

Wang H ，Falck JR ，Hall TM ，Shears SB ... -  《-》