G¦ activates PIP2 hydrolysis by recruiting and orienting PLC on the membrane surface

Phospholipase C-beta s (PLC beta s) catalyze the hydrolysis of phosphatidylinositol 4, 5-bisphosphate (PIP2) into inositoltriphosphate (IP3) and diacylglycerol (DAG). PIP2 regulates the activity of many membrane proteins, while IP3 and DAG lead to increased intracellular Ca2+ levels and activate protein kinase C, respectively. PLC beta s are regulated by G protein-coupled receptors through direct interaction with G alpha(q) and G beta gamma and are aqueous-soluble enzymes that must bind to the cell membrane to act on their lipid substrate. This study addresses the mechanism by which G beta gamma activates PLC beta 3. We show that PLC beta 3 functions as a slow Michaelis-Menten enzyme (k(cat) similar to 2 s(-1), K-M similar to 0.43 mol %) on membrane surfaces. We used membrane partitioning experiments to study the solution-membrane localization equilibrium of PLC beta 3. Its partition coefficient is such that only a small quantity of PLC beta 3 exists in the membrane in the absence of G beta gamma. When G beta gamma is present, equilibrium binding on the membrane surface increases PLC beta 3 in the membrane, increasing V-max in proportion. Atomic structures on membrane vesicle surfaces show that two G beta gamma anchor PLC beta 3 with its catalytic site oriented toward the membrane surface. Taken together, the enzyme kinetic, membrane partitioning, and structural data show that G beta gamma activates PLC beta by increasing its concentration on the membrane surface and orienting its catalytic core to engage PIP2. This principle of activation explains rapid stimulated catalysis with low background activity, which is essential to the biological processes mediated by PIP2, IP3, and DAG.