Cyclic phosphodiesterase, substrates

Other substrates for spleen exonuclease are the p-nitrophenyl esters of nucleoside-3 -phosphates and bis(p-nitrophenyl) phosphate, which is split only very slowly. These substrates are also split by enzymes having quite different natural substrates (Table I) (80-87). In fact, not only phosphodiesterases, in a broad sense, such as acid DNase, micrococcal nuclease, spleen and venom exonucleases, and cyclic phosphodiesterase but also enzymes such as nucleoside phosphoacyl hydrolase and nucleoside polyphosphatase split these substrates. As pointed out by Spahr and Gesteland (86), this may be explained by the fact that these substrates are not true diesters but rather mixed phosphoanhydrides because of the acidic character of the phenolic OH. It is evident that the use of the synthetic substrates, advocated by Razzell (3) as specific substrates for exonucleases, may be very misleading. Table II shows the distinctive characters of three spleen enzymes active on bis(p-nitrophenyl) phosphate which are present in the crude extracts from which acid exonuclease is prepared. 

The reaction mixture contained cyclic formycin monophosphate, an analog of cAMP, as the substrate, Tris-HCl (pH 7.5) as buffer, and MgQ2. The reaction was started by the addition of the enzyme. Samples were removed at intervals and injected directly onto the reversed-phase column for analysis. Figure 9.108 shows chromatograms after 10 and 30 minutes of incubation. While the amount of cFoMP substrate in the incubation mixture has declined and the amount of product FoMP has increased, the amount of formycin A (FoA), the analog of adenosine, has remained unchanged. When the area of each peak is plotted as a function of reaction time, the data shown in the central inset are obtained. Although these data clearly illustrate the activity of the cyclic phosphodiesterase, they also show the absence of any 5 -nucleotidase. 

Other examples of crossover activity are known. ASA has been shown to possess cyclic phosphodiesterase activity. Adenylate kinase transfers the 7-phosphoryl group of ATP, but can also accept as a substrate the sulfuryl analogue 7-sulfiiryl-ADP. Sulfoenolpyruvate, the sulfate analogue of phosphoenolpyruvate, is a substrate for pyruvate kinase, producing pyruvate and adenosine-5 -sulfatopyrophosphate. Many other examples probably remain undiscovered. 

When the hormonal stimulus stops, the intracellular actions of cAMP are terminated by an elaborate series of enzymes. cAMP-stimulated phosphorylation of enzyme substrates is rapidly reversed by a diverse group of specific and nonspecific phosphatases. cAMP itself is degraded to 5 -AMP by several cyclic nucleotide phosphodiesterases (PDE Figure 2-13). Competitive inhibition of cAMP degradation is one way caffeine, theophylline, and other methylxanthines produce their effects (see Chapter 20). 

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). 

FAD is cleaved by an FAD-adenosine monophosphate (AMP) lyase in liver to yield AMP and riboflavin 4, 5 -cyclic phosphate it is not known whether this has any coenzyme or cell signaling function, but it is a substrate for phosphodiesterase and has also been identified in small amounts in yeast (Fraiz et al., 1998 Cabezas et al., 2001). 

Cyclic AMP is catabolized to 5 -adenosine monophosphate by the enzyme cyclic nucleotide phosphodiesterase, which terminates any further cAMP-initiated reactions. This enzyme also requires Mg + for activity. Calcium, again in consort with calmodulin, can stimulate phosphodiesterase activity. Phosphodiesterase appears to exist in multiple forms, each with specificity toward different substrates. Calcium and calmodulin activate only one form of the enzyme. The enzyme is potently inhibited by methyl xanthines, such as caffeine, theophylline, and theobromine. It is believed that at least part of the pharmacological effects of such compounds can be explained through their inhibition of phosphodiesterase and the consequent reduction in the catabolism of cAMP. 

The first application of this 0 effect for determining the configuration of an oxygen chiral phosphate ester was the author s determination of the configuration of diastereomeric samples of cyclic [, 0]dAMP, the chiral substrate for studying the stereochemical consequences of the reverse reaction catalyzed by adenylate cyclase (formation of cyclic AMP from ATP), and of the hydrolysis reaction catalyzed by 3, 5 -cyclic nucleotide phosphodiesterase (25) (see Fig. 

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