Phospholipase C enzyme
- Phospholipase C (PLC) enzymes are responsible for the hydrolysis of the inner membrane component phosphatidylinositol 4, 5 bisphosphate (PIP2) to the second messengers inositol-1,4,5-triphosphate (IP3) and diacylglycerol (DAG).
- IP3 is freely diffusible and binds to IP3-specific receptors, leading to the release of intracellular Ca2+.
- DAG remains membrane associated and together with increasing Ca2+it activates protein kinase C.
- These events are associated with the regulation of numerous physiological processes:
(Muscle contraction, chemotaxis, opioid sensitivity, cell proliferation and survival)
- There are six subfamilies of PLC in higher eukaryotes.
- PLC β subfamily enzymes are the downstream targets of the Gq subfamily of G protein coupled receptors (GPCRs) and play prominent roles in cardiovascular function, chemotaxis, and neuronal signaling.
- PLC β activated upon direct interactions with Gαq.
- GPCR mediated activation of PLC β also occurs through release of the G βγ heterodimer, which is thought to be mediated by activation of Gi coupled GPCRs
- Members of the Rho family of small molecular weight G proteins, such as the Rac isoforms, also directly bind and activate PLC β (linking PLC β activity to GPCR independent signaling cascades)
- In mammals, PLCs share a conserved core structure and differ in other domains specific for each family.
- The beta isoform of phospholipase C has an additional domain at its carboxyl terminus. This domain interacts with α subunit of Gq in its GTP bound form.
PLC-β forms: Activated by heterotrimeric G proteins (either their α or βγ subunits)
PLC-γ forms: Regulated by receptor tyrosine kinases.
These two receptors activate the isoforms of phospholipase C through various mechanisms.
The core enzyme includes:
Triosephosphate isomerase (TIM) barrel:
- The TIM barrel contains the active site, all catalytic residues, and a Ca2+binding site.
- It has an auto inhibitory insert that interrupts its activity called an X-Y linker. When it is removed PLC is activated.
Pleckstrin homology (PH) domain:
- This ~120-residue domain binds a lipid head group such as that of PIP2.
- PH domains facilitate the binding of proteins to membrane lipids particularly PIP2.
- PH domains help to localize the enzyme phospholipase C near its substrate PIP2.
- The PH domain is joined to the catalytic domain by a set of four EF-hand domains.
- This ~130-residue domain is a member of the immunoglobulin domain super family and plays a role in binding phospholipid head groups.
The PH and C2 domains of phospholipase help to position the enzyme’s catalytic site for ready access to the phosphodiester bond of the membrane lipid substrate PIP2.
EF Hand domain:
- Formed by a helix-loop-helix unit. EF hand is a binding site for calcium in many calcium sensing proteins.
- PLC have Four tandem EF hand domains
- PLCs perform their catalytic function at the plasma membrane where their substrate PIP2 is present.
- This membrane docking is mediated mostly by lipid-binding domains (e.g. PH domain and C2 domain).
Catalytic action of enzyme
Phospholipase C (PLC) is a class of membrane associated enzymes that cleave phospholipids just before the phosphate group.
Cleavage site: hydrolyzes the phosphodiester bond linking the phosphorylated inositol unit to the acylated glycerol moiety.
Phospholipase C catalyses the hydrolysis of PIP2 (phosphatidylinositol 4, 5 bisphosphate (PIP2) into DAG (Diacyl glycerol) and IP3 (inositol-1,4,5-triphosphate).