Phosphodiesterases, from snake venom and

That the phosphorus is pi esent as a monoester is further supported by the finding that on pretreatment of ovalbumin with the phosphodiesterase from snake venom and subsequent incubation at pH 5.6 with prostate phosphatase, the same amount of phosphorus is released as with this enzyme alone, i.e., 46%. Moreover, no change in the electrophoretic behavior occurs (72). 

A uridine phosphate (5), obtained by treatment of uridine 5 -(a-D-glucopyranosyl pyrophosphate) with ammonia, imdergoes hydrazinolysis to D-ribose 5-phosphate (2) and 3-pyrazolone, which establishes the structure of (S) as uridine 5 -phosphate. - (Hydrazinolysis of uridine to pyrazolone, with the liberation of the sugar moiety, had been described, and has served as a useful tool in the determination of the position of attachment of the sugar moiety to the aglycon. ) The 5 -phosphates of adenosine, guanosine, cytidine, and uridine were obtained by enzymic hydrolysis of ribonucleic acid with phosphodiesterases from snake venom and from other sources (such as Streptomyces aureu ). 

E. Phosphodiesterases from Snake Venom and Bovine Spleen. 133... 

The stereochemical consequences of the reactions catalyzed by these two phosphodiesterases are the opposite of those determined for the reactions catalyzed by SNase and DNase I. The structural and mechanistic divergence already noted for the two phosphodiesterases, which require Ca for activity and probably involve general base-assisted attack of water on the substrate, is expanded on by the finding that the phosphodiesterases from snake venom and spleen utilize mechanisms that have retention as their stereochemical outcomes. Clearly, the stereochemical and structural studies of phosphodiesterases reported to date reveal mechanistic complexity that contrasts with the stereochemical (and mechanistic) uniformity that has been discovered for all of the kinases studied to date. The diversity in the phosphodiesterases is perhaps even more surprising when it is realized that the substrates for all of the enzymes that have been stereochemi-... 

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... 

We have also studied the stereochemical outcomes of the reactions catalyzed by the phosphodiesterase from snake venom (99) and from bovine spleen (101). The former enzyme catalyzes the hydrolysis of esters of 5 -nucleotides (free 3 -hydroxyl group), and the latter enzyme catalyzes the hydrolysis of esters of 3 -nucleotides (free 5 -hydroxyl group). No structural information of mechanistic consequence is available for these enzymes. 

Both mechanisms account for the observed inversion of stereochemistry. The second mechanism proceeds by an adjacent displacement occurring with retention, followed by an in-line displacement with inversion. This doubledisplacement mechanism requires a pseudorotation at phosphorus, the intermediacy of multiple TBP intermediates, and the intermediacy of a covalent phosphoryl enzyme intermediate. An adenylated enzyme has been isolated in poor yield from a phosphodiesterase capable of hydrolysing cAMP (Landt and Butler, 1978), and the phosphodiesterase from snake venom has been shown to operate via a double-displacement mechanism (Burgers et al., 1979a). Eckstein points out that the choice between the two proposed mechanisms is dependent (i) on the conformational preference of a six-membered ring in a TBP species, and (ii) on proof for a two-step mechanism for cAMP phosphodiesterase. 

Bryant, F. R. and Benkovic, S, J. (1979) Stereochemical course of the reaction catalyzed by nucleotide phosphodiesterase from snake venom. Biochemistry 18, 2825-2828... 

A snake venom is a rich source of enzymes. Many enzymes are purified from snake venoms and sold commercially. These include phosphodiesterase, L-amino acid oxidase, 5 -nucleotidase, thrombin-like enzymes (ancrod, atroxin, crotalase, reptilase), and thrombo-cytin. 

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

The third type of enzyme presumably involved in degradation of the polymer is phosphodiesterase. The snake venom enzyme has been used extensively to degrade the polymer for structural anedysis. The nuclear phosphodiesterase from liver will hydrolyze the pyrophosphate bonds of poly(ADP-ribose), NAD, NADH, and ADP-ribose 135). Futai has demonstrated that the hydrolysis of poly( ADP-ribose) proceeds from the AMP terminus 71) (refer to Fig. 4). 

Venom Phosphodiesterase. A phosphodiesterase from the venom of several species of snakes exhibits extreme specificity with regard to nucleotides. It hydrolyzes only components from a phosphate esterified at a 5 position. Thus, 5 nucleotides are liberated from RNA. The venom diesterase attacks both purine and pyrimidine nucleotides (Fig. 26). This activity was useful in establishing that 3 -5 linkages, rather than 2 -3 linkages, occur in nucleic acids. A similar enzyme occurs in intestines. 

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

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... 

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. 

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... 

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. 

Stec applied the above-described oxathiaphospholane approach to synthesise stereoselectively the phosphorothioates of the locked nucleic acids (84a) and (84b) from (83a) and (83b), respectively (Scheme 7). The oxathiaphospholane ring opening condensation reaction proceeded in acetonitrile in high yield and with 96% stereoselectivity. One of the two diastereomers thus prepared was found to be readily digested by snake venom phosphodiesterase, an enzyme known to be an Kp-specific nuclease. However, neither diastereoisomer was hydrolysed by nuclease PI, an enzyme known to preferentially hydrolyse phosphorothioate linkages of Sp-configuration. 

The reaction of 5 -amino-5 -deoxyadenosine with trimetaphosphate affords the 5 -Af-triphosphate (23). When (23) is employed as substrate with glucose in the hexokinase-catalysed reaction, the 5 -AT-diphosphate (24) is obtained the latter is cleaved by snake venom phosphodiesterase to the 5 -phosphoramidate, and hydrolyses in acid to the amino-nucleoside. It does not appear to be polymerized by polynucleotide phosphorylase. In this context it is noteworthy that uridine 5 -5-thiopyrophosphate (25) is a competitive inhibitor for polynucleotide phosphorylase from E. coli, but not a substrate, and that the 5 -S-thiotriphosphates (26) and (27) show neither substrate nor inhibitory properties for RNA polymerase or DNA polymerase I, respectively. However, (23) can be polymerized using the latter enzyme, showing that the introduction of a 5 -heteroatom does not completely exclude these modified nucleotides as substrates for the polymerizing enzymes. 

Nucleotides in aqueous solution can be alkylated at the phosphate (and in some cases the nucleoside also) by the action of l-oxidopyridin-2-yldiazo-methane (33). - The protecting group may be removed from the phosphate with snake venom phosphodiesterase, or generally by acetic anhydride treatment, followed by ammonia. Phosphoramidates have been described previously as phosphate-protecting groups, and if 2-naphthylamine is used as its anilidate for this purpose, organic solvent extraction (as above) is possible. A variation on this theme is to use dianilidophosphochloridate (34) as a... 

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