Phosphodiesterase probes

Another important group of hydrolytic enzymes are phospho- and cyclophosphodiesterases. They catalyze the hydrolysis of phospho-diester bonds and many of the most relevant biological substrates are nucleic acids. Phospholipase C and D are also important examples. Initial attempts to measure phosphodiesterase activity placed a phosphodiester between a fluorophore and a quencher and the probe was tested in vitro [146], This system was slightly modified by Caturla and used for the identification of catalysts with phosphodiesterase activity [147], More recently, Nagano and co-workers used a coumarin donor and fluorescein as a FRET... 

Fig. 6.18. Design and performance of the FRET probe CPF4 for measuring phosphodiesterase activity. Ri and R2 are phosphate and hydrogen or vice versa.

Caturla, F., Enjo, J., Bemabeu, M. C. and Le Serre, S. (2004). New fluorescent probes for testing combinatorial catalysts with phosphodiesterase and esterase activities. Tetrahedron 60, 1903-1911. 

GTP-bound a-subunit activates a cGMP-specific phosphodiesterase as described briefly earlier. A benzophenone derivative of cGMP was prepared and found to inhibit several types of cyclic nucleotide phosphodiesterases at low concentrations. In rod outer segment preparations, the [a-32P]-tagged probe... 

Since it is well established that the P-chiral phosphorothio-ates serve as effective probes in mechanistic studies for the phos-phoryl group transfer enzymes /16/, we turned our attention to the application of diastereomeric 8 (B=Thy, Ar=pN02C6Hi -,/17f) to elucidate the mode of action of spleen phosphodiesterase (SPDE, EC 3.1.1. 18). This enzyme splits the phosphodiester bonds to yield nucleoside 3 -phosphates. In the case of it was expected that its SPDE-catalyzed hydrolysis in 180 H 2O medium leads to P-chiral thymidine 3 - 180 phosphorothioate. On the contrary to our expectation the main product of this reaction was thymidine cyclic 3 ,5 - Rp phosphorothioate (10) /6/. By treatment of 8 under the same conditions, but in the absence of the enzyme, no trace of J 0 was detected. ... 

For all the PI-PLC enzymes examined thus far, PI hydrolysis occurs in two sequential reactions as shown in Figure 7 (Griffith and Ryan 1999) (i) an intramolecular phosphotransferase reaction at a phospholipid/aggregate surface to produce DAG and water-soluble 1,2-cyclic phosphate, cIP (or cIPx in the case of a phosphoinositide substrate), followed by (ii) a phosphodiesterase reaction where the cIP is hydrolyzed to inositol-1 -phosphate (or IPX). The second reaction can be studied separately it occurs with a soluble monomeric substrate and is much slower (both lower kcat and higher Km) than the first step (Volwerk et al., 1990 Zhou et al., 1997). It is an excellent probe for allosteric effects of membranes on catalysis (Zhou et al., 1997 Wu et al., 1997). 

A novel fluorescent probe, TG-mPC (35), which could selectively detect nucleotide NPP6/lysophospholipase C (choline-specific glycerophos-phodiester phosphodiesterase) has been developed by Nagano et al ... 

---