Snake venom phosphodiesterases

The question of enzyme specificity for irradiated polynucleotides is taken up in more detail in the recent review of Johns.11 The specificities of four enzymes, spleen phosphodiesterase, snake venom phosphodiesterase, pancreatic ribonuclease, and pancreatic deoxyribonuclease are discussed. 

Thiamine derivatives inhibit glucose synthesis, transketolase (yeast), phosphodiesterase (snake venom), thiamine triphosphatase, thymidylate kinase. 

Ribonucleic Acid. A major contribution to the formulation of RNA structure was the demonstration that alkaline hydrolysis of RNA quantitatively liberates about equal amounts of mononucleotide isomers of all four bases 103). Although it was readily established that none of these mononucleotides is the 5 -phosphate isomer, it was not until some years later that Cohn and associates 103) by controlled degradation experiments, and Brown and associates 121) by the synthetic route, established that the products were isomers involving phosphate attachment at positions 2 and 3 of the ribose. Of equal significance was the discovery 161) that hydrolysis of RNA by the enzyme phosphodiesterase (snake venom or intestinal) liberates mononucleotides exclusively of still another type, the 5 -mono-nucleotides. It was thus necessary to establish the mechanisms which could account for one phosphodiester structure in the RNA chain giving rise to three isomers of each mononucleotide. 

Furthermore, incubation of APh-GDP with alkaline phosphatase (intestinal) and phosphodiesterase (snake venom) liberates 4-azidophenol. This leads to an increase in absorption at 303 nm. Incubation with phosphodiesterase alone produces APh-P, and incubation with alkaline phosphatase alone is without effect. 

B. Phosphodiesterase. Snake venoms commonly contain enzymes that hydrolyze phosphodiester bonds. There are two types of phosphodiesterase one is an exonuclease and the second is an endonuclease. 

FIGURE 11.31 Snake venom phosphodiesterase and spleen phosphodiesterase are exonncleases that degrade polynncleotides from opposite ends. 

Snake venom phosphodiesterase Both a Starts at 3 -end, 5 -NMP products... 

Ribonucleoside 5 -0-hydroxymethylphosphonates (8 R = OH) are resistant to the action of phosphatases and phosphodiesterases. They are, however, good substrates for snake venom 5 -nucleotidase, unlike (8 R = H).2 ... 

Phosphoramidate analogues of dideoxyribonucleoside phosphates (26) and trideoxyribonucleoside phosphates are acid labile and can be hydrolysed enzymically. Snake venom phosphodiesterase cleaves (26) to thymidine and 5 -deoxy-5 -aminothymidine (27 R = H). The latter presumably arises by spontaneous decomposition of the phosphoramidate (27 R = PO3H2) and P—O fission must have occurred during the initial hydrolysis. With acid or spleen phosphodiesterase, (26) gave Tp and (27 R = H), i.e. P—N fission occurred. 

Using phosphotriester methods, dinucleoside (3 - 50-monophosphates containing 6-methyl-2,-deoxyuridine at the 3 - or 5 -end have been prepared.44 N.m.r. spectroscopy indicates that this nucleoside possesses the syn conformation in these compounds, and, on treatment with snake venom phosphodiesterase, d(m6UpT) is degraded, while d(Apm6U) is not, indicating that this enzyme, a 3 -exonuclease, requires the anti conformation to be present in the substrate. Two modified nucleo-side-5 -monophosphates, (20) and (21), which are resistant to 5 -nucleotidase, have been isolated from tRNA snake venom hydrolysates.45 A synthesis of (20) has been reported.46... 

The existence of photoreversible, but not of heat-reversible, absorbance change in irradiated poly dI dC was taken to prove that the photoproducts are entirely dimers (in contrast to those in poly C irradiations where the product is almost entirely the hydrate82a). It was possible to detect dimers of uracil as well as those of cytosine, by means of the much slower photoreversal of uracil dimers. In the acid hydrolysates of irradiated dl-dC, both uracil dimers and uracil could be identified. Enzymatic hydrolysis (snake venom phosphodiesterase) does not split pyrimidine dimers, and the products of such hydrolysis of irradiated tritium-labeled poly dl dC contained trinucleotides shown by radioactivity to contain cytosine dimers. Thymine dimers were formed in the photolysis of the poly dA dT, and were detected and assayed by the same methods. The yield of thymine dimers in irradiated poly... 

Apart from important similarities in the endo- and exonucleolytic properties of staphylococcal nuclease and other well-studied phosphodiesterases (67), those from snake venom and spleen, the basic structural substrate elements for these enzymes appear to be quite different... 

Fig. 2. Proposed structural requirements for substrates of phosphodiesterases that hydrolyze DNA and RNA, those from (a) snake venom, (b) spleen, and (c) staphylococcus (R = thymine and R — p-nitrophenyl). The studies indicated for the venom and spleen enzymes are those suggested by Khorana (67) [data from Cuatrecasas el al. (61)).

In addition to the proposed regulatory role of ATP and pyrophosphate, some possibility exists that 3, 5 -cyclic phosphate diesterase is under physiological control. Such ideas arose through observations of Cheung (43, 62) that the partially purified enzyme from beef brain was markedly activated by snake venom. The stimulatory factor was labile at extreme pH it was not dialyzable and appeared to be a protein. A similar activating factor is also present in brain tissue (63) and is removed during purification of the diesterase. It seems to interact stoichiometrically with the enzyme. The activator is destroyed by trypsin and is not proteolytic itself. The precise role of this protein in regulating the phosphodiesterase in vivo is not yet established, however. 

When the Tg lesions is opened by ammonolysis, the resulting product (ureidoisobutyric acid) inhibits snake venom phosphodiesterase, A exonuclease and the Klenow (exo ) fragment (Matray et al. 1995 see also Greenberg and Matray 1997). It is, however, removed by E. coli Fpg and Nth proteins (Jurado et al. 1998). 

All three of the fully-unblocked dodecamers underwent complete digestion to give their monomeric components when they were treated with Crotalus adamanteus snake venom and spleen phosphodiesterases. Their structures were further confirmed in the usual way. 

SVPDE/AP snake venom phosphodiesterase and alkaline phosphatase t-BOC tm-butoxycarbonyl (group)... 

Snake Venom Phosphodiesterase An exonuclease is an enzyme that sequentially cleaves nucleotides from the end of a polynucleotide strand. Snake venom phosphodiesterase, which hydrolyzes nucleotides from the 3 end of any oligonucleotide with a free 3 -hydroxyl group, cleaves between the 3 hydroxyl of the ribose or deoxyribose and the phosphoryl group of the next nucleotide. It acts on single-stranded DNA or RNA and has no base specificity. This enzyme was used in sequence... 

Phosphodiesterase I Snake venom RNA and DNA exonudease splits p bonds from 3 ends, releasing 5 -phosphonucleosides... 

Iqbal et al. studied the phosphodiesterase inhibitory effect of durantins A (33), B (15), and C (16), isolated from Duranta repens (70). Compounds 16 and 33, along with compound 9, showed moderate to strong inhibitory activity against snake venom phosphodiesterase 1, using cysteine and EDTA as positive controls, while 15 showed weak activity. 

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