Phosphoinositide-specific phospholipase C delta1 activity toward micellar substrates, inositol 1,2-cyclic phosphate, and other water-soluble substrates: a sequential mechanism and allosteric activation.
The kinetics of full-length and PH domain truncated cloned PI-PLC delta1 from rat toward soluble substrates [inositol 1, 2-(cyclic)-phosphate (cIP) and glycerophosphoinositol phosphates (GPIPx)] as well as PI in detergent micelles provide the following insights into the mechanism of this enzyme. (i) That cIP is a substrate for the enzyme implies a two-step mechanism for PI hydrolysis [intramolecular phosphotransferase reaction to form cIP followed by cyclic phosphodiesterase activity to form inositol-1-phosphate (I-1-P)]. The dependence of enzyme activity on cIP is sigmoidal, suggesting a transition between less active and more active forms of the enzyme that is affected by substrate. (ii) Interfaces increase the kcat for cIP (but do not affect the cooperativity), and this allosteric activation requires an intact PH domain. (iii) Phosphorylation of the soluble inositol phosphodiesters GPI, GPIP, and GPIP2 enhances PI-PLC delta1 activity by dramatically increasing kcat and decreasing Km. For these phosphodiesters, the substrate saturation curve is no longer sigmoidal but hyperbolic, indicating the phosphorylated substrate can shift the enzyme to the activated form. (iv) Given the kinetic parameters for cIP hydrolysis and the constant ratio of cIP/I-1-P generated during PI hydrolysis, the cIP produced in situ is either released (and not readily rebound since its concentration is well below Km) or attacked by a water molecule for the generation of the acyclic product.
Wu
Y
Y
Merkert Chemistry Center, Boston College, Chestnut Hill, Massachusetts 02167, USA.
Perisic
O
O
Williams
R L
RL
Katan
M
M
Roberts
M F
MF
D000494:Allosteric Regulation; D000818:Animals; D006868:Hydrolysis; D007295:Inositol Phosphates / Q000378:metabolism*; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D008823:Micelles; D008956:Models, Chemical; D017830:Octoxynol; D010716:Phosphatidylinositols / Q000378:metabolism*; D054803:Phospholipase C delta; D051381:Rats; D012995:Solubility; D010738:Type C Phospholipases / Q000378:metabolism*; D014867:Water
D007295:Inositol Phosphates; D007527:Isoenzymes; D008823:Micelles; D010716:Phosphatidylinositols; D014867:Water; C010199:inositol cyclic phosphate; D017830:Octoxynol; D010738:Type C Phospholipases; D054803:Phospholipase C delta; C518026:Plcd1 protein, rat