Venom exonuclease substrates

Venom exonuclease [EC 3.1.15.1], also known as venom phosphodiesterase, catalyzes the exonucleolytic cleavage of RNA or DNA (preferring single-stranded substrates) in the 3 to 5 direction to yield 5 -phosphomononucleo-tides. Similar enzymes include hog kidney phosphodiesterase and the Lactobacillus exonuclease. See also specific phosphodiesterase J. A. Gerit (1992) The Enzymes, 3rd ed., 20, 95. 

In spite of some claims to the contrary, venom exonuclease is capable of attacking double-stranded high molecular DNA. In fact, double-stranded DNA is a better substrate than denatured DNA. Bjork (1 ) studied the rates of degradation of native and heat-denatured DNA using a pH stat. Denatured DNA was degraded at a steady rate, which was dependent on the ionic strength of the medium. An increase in NaCl concentration from 1 to 100 mM decreased the rate of hydrolysis by a factor of two. With native DNA a two-phase reaction was observed. The initial, very rapid, rate was independent of NaCl concentration. After about one-third of the linkages had been hydrolyzed, the rate slowed down to that of denatured DNA and became salt dependent. Similar biphasic kinetics was observed previously with DNA that was denatured by an exhaustive dialysis (40). 

A good source of uncommon bases is tRNA. It provides substrates for studying the effect of base on the rate of hydrolysis. Baev et al. (62) showed that V2-dimethylguanylyl-(3 -5 )-cytidine-3 phosphate (G2m-pCp) was hydrolyzed much slower than the usual GpCp. Venkstern (63) reported that Tp was hydrolyzed very slowly. Naylor et al. (64) found that Cp was hydrolyzed with half the rate of CpU. The same group of workers introduced (64, 65) a chemical block on uridine and pseudo-uridine residues by reacting RNA with l-cyclohexyl-3-(2-morpho-liny]-(4)-ethyl)-carbodiimide metho-p-toluene sulfonate. The modification of the uridine residues completely blocked the action of venom exonuclease and also blocked the action of pancreatic RNase. 

Derivatives bearing a 3 -monopbosphoryl group were originally classified as totally resistant to venom exonuclease. As the quality of the enzyme preparation improved, these compounds were found susceptible but required 1000-fold more enzyme than was needed to hydrolyze 5 -monophosphate-bearing compounds. This unusual resistance led to another erroneous conclusion, that the polarity of exonuclease changes (20). The basis for this belief were the experiments in which a mixture of tri-, tetra-, and pentanucleotides of the type d-N pNPpN pN p were used as substrates. The early products were nucleosides and nucleotides, whereas 3, 5 -mononucleoside diphosphates appeared considerably later. It is clear now that the mixture was contaminated with a small amount of dephosphorylated chains which were rapidly hydrolyzed to completion. 

Fig. 3. Effect of pH on the hydrolysis of purified mixed deoxyribotrinucleotides (d-N pNspN1 p) by venom exonuclease. (A) d-pN p, (X) d-N pN /2, ( ) d-pN, and (O) d-N . Unhydrolyzed d-N pN pN1 p is not shown but is included in the total Am. Substrate = 83 A271, enzyme] = 0.525 unit/ml for pH 4.0, 0.210 unit/ml for pH 5.0 and 9, and 0.105 unit/ml for pH 6-8. Electrophoresis for 2 hr, pH 6. Observed values for (percent of total A27i) of the separated products were divided by 5.2 or 1 to compensate for the different amounts of enzyme used. Dashed line shows values for mononucleotides without such correction. Values of percent of total Am for d-N"pN were divided by 2 to facilitate comparison with other products on a molar basis. Reprinted from Richards and Laskowski (89). Copyright (1969) by the American Chemical Society. Reprinted by permission of the copyright owner.

As the quality of venom exonuclease improved, more difficult tasks were tackled. The a terminus was identified in tobacco mosaic virus (72). It required finding one nucleoside among 6400 nucleotides. Venom exonuclease was also used for identification of both terminals in chains bearing 3 -monophosphate (IS, 73). The a terminus appears as a nucleoside, the wide application because it required large amounts of highly purified enzyme. The recent finding that 3 -monophosphates are better substrates at pH 6 than 9 (29) is likely to increase the use of this method. 

In passing, it may be mentioned that m-exonuclease activity presumably similar to venom exonuclease has been studied histochemically in a variety of tissues. Several substrates have been developed specifically for this purpose (76-79). The newest addition to the family of 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 ATPase of most biological systems hydrolyzes the a-phosphate producing ADP + Pj. When ATP is used as a substrate, snake venom exonuclease hydrolyzes it to AMP and PP (pyrophosphate). However, when ATP is mixed with snake venom, it is hydrolyzed to adenosine, Pj and PP because snake venom contains an additional enzyme, 5 -nucleotidase. This can be summarized as follows ... 

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... 

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