Phospholipase catalytic mechanism

A consistent catalytic mechanism of phospholipase C from Bacillus cereus based on molecular mechanics calculations was reported by da Graja Thrige and coworkers . The... 

Crystal structure of human cytosolic phospholipase A2 reveals a novel topology and catalytic mechanism. Cell 97 349-360. 

Phospholipases 2 are found in high concentrations in the venom of reptiles and invertebrates. These enzymes are probably similar to the pancreatic phospholipase 2 in structure and catalytic mechanism. They break down cellular membranes in the victim s tissues by hydrolysis of the phospholipids and thus assist in spreading the toxin. 

Dijkstra, B. W., Drenth, J., and Kalk, K. H. (1981a). Active site and catalytic mechanism of phospholipase A2. Nature (London) 289,604—606. 

Figure 17 Proposed catalytic mechanism for phospholipase C. Zinc ions are indicated as filled circles (reproduced by permission of Springer from Flergenrother and Martin ).

Figure 6.3. Mechanism of action of heterotrimeric G-proteins. Upon receptor occupancy, the Ga-subunit binds GTP in exchange for GDP, and then moves in the membrane until it encounters its target enzyme, shown here as adenylate cyclase (alternatively, a phospholipase). The activated target enzyme then becomes functional. Inherent GTPase activity within the a-subunit then hydrolyses bound GTP to GDP, and the a-subunit dissociates from its target enzyme (which becomes inactive) and rebinds the / - and ysubunits. Upon continued receptor occupancy, further catalytic cycles of GTP exchange and target enzyme activation may occur. The scheme shown is for a stimulatory G-protein (Got,), but similar sequences of events occur with inhibitory G-proteins (Gcx,) except that the interaction of the a-subunit with adenylate cyclase will result in its inhibition. The sites of action of pertussis and cholera toxins are shown.

Several cases are described (Cohen et ah, 1995) in which binding of an SH2-containing enzyme to an activated receptor tyrosine kinase leads to increased catalytic activity of the enzyme. Examples are the PI3-kinase and phospholipase Oy. The mechanism of activation is not clear. It is possible, however, that the basis is an allosteric mechanism, as is assumed for activation of Src tyrosine kinase. The Src kinase can be phosphoryla-... 

Schiavo G, Papini E, Genna G, Montecucco C (1990) An intact interchain disulfide bond is required for the neurotoxicity of tetanus toxin. Infect Immun 58 4136 11 Scott AB, Magoon EH, McNeer KW, Stager DR (1989) Botulinum treatment of strabismus in children. Trans Am Ophthalmol Soc 87 174-180 discussion 180 1 Scott D (1997) Phospholipase A2 structure and catalytic properties. In Kini R (ed) Venom phospholipase A2 enzymes structure, function and mechanism. John Wiley Sons, Chichester, p 97-128. 

Having shown that dibutyryl PC is monomeric under the enzyme assay conditions, we found that the phospholipase A2, which acts poorly on PE in mixed micelles, is activated by dibutyryl PC which is itself an even poorer substrate. 31p-NMR spectroscopy was employed to show that only PE is hydrolyzed in mixtures of various compositions of these two phospholipids. The fully activated enzyme hydrolyzes PE at a similar rate to its optimal substrate, PC containing long-chain fatty acid groups. Because dibutyryl PC is not incorporated into the micelles, these results are consistent with a mechanism of direct activation of the enzyme by phosphoryl-choline-containing lipids (either monomeric or micellar) rather than a change in the properties of the interface being responsible for the activation of phospholipase A2. Therefore, two functional sites on the enzyme have to be assumed an activator site and a catalytic site (6). 

It remains unknown how GTP-bound a or py of G-proteins interacts with the effector system such as phospholipase C or ion chaimels. In the case of adenylate cyclase, the activation involves direct interaction of the GTP-bound a-subunit of G s with the adenylate cyclase catalyst. The inhibition of adenylate cyclase, however, results from combined effects of direct and indirect interactions between some a-subunits, Py -subunits and the catalytic protein of adenylate cyclase as follows. Firstly, Py liberated from Gi, Go and Go may form a trimeric complex with the a-subunit of Gs, thereby decreasing the concentration of free as, the direct activator of the cyclase catalyst (31). The inhibition by this mechanism could be expected to occur in a number of mammalian cell types, since these lAP substrates are much more abundant than Gs in these cells (37). Secondly, the a-subunit of Gi competes with the a-subunit of Gs for the activation site on the cyclase catalyst, though the affinity of Gi was much lower than the affinity of Gs for this site (38). No competition was observed, however, between the a-subunit of G s and a-subimits of Go, Go and G hl. Thirdly, py is capable of direct interaction with the adenylate cyclase catalyst in such a manner as to lower the cyclase activity (38). The interaction was observed at rather higher concentrations of Py. Fourthly, Py binds to calmodulin with a high affinity (39). Calmodulin is a potent activator of the adenylate cyclase catalyst as such, but is not so after it is bound by Py of G-proteins. Thus, the inhibition of adenylate cyclase by Py of G-proteins was biphasic in the presence of calmodulin the inhibition by lower concentrations of Py was due to prevention of calmodulin activation of the cyclase and the inhibition by the higher concentrations reflected the direct interaction with the cyclase. The relative importance of these multiple mechanisms for adenylate cyclase inhibition will be the subject of future investigations. 

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