Venom exonuclease

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. 

Velocity-modulated allosteric regulation, V, ,-TYPE ALLOSTERIC SYSTEM VENOM EXONUCLEASE Venom phosphodiesterase, PHOSPHODIESTERASES VESICLE TRANSPORT IN CELLS Vibrational bond stretching mode,... 

Venom has long been known to be a good source of several enzymes that hydrolyze esters of phosphoric acid. It is not possible to discuss venom exonuclease without mentioning other enzymes of this group. An effort will be made, however, to limit the discussion of other phosphatases to the bare essentials and key references. The surveys of different species of snake with respect to these enzymes are fairly numerous (1-9) and allow several conclusions to be drawn. 

Third, the quantitative differences in concentration of the four enzymes vary appreciably therefore making several venoms an undesirable source of exonuclease (5). All partially purified preparations of venom exonuclease exhibit adenosine triphosphate-pyrophosphatase activity (cleavage of the a-/3 linkage). Pfleiderer and Ortanderl (14a) studied this issue and showed that during purification the ratio of the two activities remains constant, concluding that both activities are intrinsic properties of the same enzyme. 

The terms venom exonuclease and venom phosphodiesterase are at present used interchangeably to designate the same enzyme. The reviewer prefers the first, because he would like to see phosphodiesterase restored to it original meaning as the general name for all enzymes attacking diesterified phosphate. During the recent past, venom exonuclease has been reviewed several times (15-21). Three books (22-24) devoted to nucleases discuss venom exonuclease. 

It is now clear that venom exonuclease attacks all polynucleotide chains from the w terminus regardless of the location of the monophos-phoryl group (see below). 

Presumably many more enzymes of this type will be found. Among the presently known nucleases only a few exhibit the w-monophosphatase activity. Venom exonuclease which belongs to the group of m-exonudeases does not have this property (see Section IV,B). 

It has been known for some time (16) that venom exonuclease produces only 5 -mononucleotides and that it attacks both deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Recently, however, it was shown that it also attacks derivatives of arabinose (30, 34, 35) and... 

Very little is known about the chemical nature and physical properties of venom exonuclease we simply do not have a preparation pure enough to warrant such studies. This regrettable state of affairs is caused in part by the high price of the starting material. No laboratory can afford a macroscale purification procedure. Therefore, the major aim up to the present time has been to obtain a preparation free from the contaminants that interfere with a specific use of the enzyme. This approach resulted in a number of preparations that varied not only in specific activity of exonuclease but also in the nature, and quantity of contaminants. The criteria used to describe these remaining contaminating activities vary. Such statements as below the level of detection are helpful only if the level is specified. Unfortunately, this is not always the case. 

Several methods for preparation of purified venom exonuclease have been described [see the reviews on methods (15, 18, 19, 21) and books (22-24)]. The major effort of purification was directed toward removing the contaminating monophosphatases. A successful and widely used step was introduced by Sinsheimer and Koerner (43). At pH 4, monophosphatases are precipitated with a lower concentration of acetone the remaining exonuclease is precipitated by a higher acetone concentration. Several modifications of this principle have been proposed (concentration of acetone, temperature during precipitation, etc.). Commercially available preparations represent essentially this stage and contain per unit of exonuclease 10 4 unit of 5 -nucleotidase, 10 3 unit of nonspecific phosphatase, and about the same amount of endonuclease. The last figure is only an approximation because of the difficulty of accurate determination. The more elaborate preparations have these contaminants... 

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). 

It has been known for many years (15-24) that venom exonuclease is capable of hydrolyzing both DNA and RNA. Gray and Lane (56) showed that naturally occurring 2 -0-methyl-substituted ribose derivatives are also hydrolyzed, even though the rate of hydrolysis is slower than with the unsubstituted ribose. Interestingly, the 2 -0-methylated derivatives are totally resistant to 5 -nucleotidase (57, 58). 

Wechter (34) and Richards et al. (35) showed that venom exonuclease is capable of hydrolyzing dinucleoside monophosphates w ith one or both nucleosides containing arabinose. Even though only four different sugars have been tested, it would appear that venom exonuclease is totally blind to sugar, at least in the qualitative sense. Other enzymes capable of hydrolyzing DNA and RNA are incapable of hydrolyzing derivatives of arabinose, e.g., micrococcal nuclease (SO). 

Quantitative differences between susceptibility of compounds containing different sugars exist. Richards et al. (35) made a systematic study of a number of dinucleosidemonophosphates (N°pNP) in which either the a- or /3-nucleoside contained arabinose, while the other nucleoside contained ribose or deoxyribose. All compounds were found susceptible to venom exonuclease. The surprising finding was that the extremes of differences in the values of Vm and Km lay in a comparatively narrow range of 20-fold. 

Venom exonuclease has no pronounced preference (or discrimination) toward any of the four common bases of DNA or RNA. The discrimina-... 

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. 

The action of venom exonuclease is also blocked by thymine dimers produced as a result of UV irradiation of DNA at 280 nm. Setlow et al. (66) subjected irradiated DNA to exhaustive digestion by venom exonuclease. They isolated and identified the products of the reaction, which were composed of large amounts of all four 5 -mononucleotides and of small amounts of trinucleotides of the type d-pNpTpT where N was any of four common nucleosides and TpT was the irradiation-induced dimer of thymidine. These trinucleotides were totally resistant to further digestion with venom exonuclease but became partially susceptible after UV irradiation at 240 nm, known to reverse dimerization. The authors picture the action of venom exonuclease as proceeding linearly from the internal bond one base beyond the dimer. From there on conventional hydrolysis is resumed until the next block. This experiment touches upon one of the most pressing problems connected with venom exonuclease. Is the endonucleolytic activity an intrinsic property of the enzyme ... 

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.

Venom exonuclease has been widely used for the determination of the a terminus in oligonucleotides. The normal procedure was to divide the dephosphorylated sample into two parts and digest one part with venom exonuclease. The a terminus appeared as a nucleoside the rest of the chain was degraded to 5 -mononucleotides. The other part of the dephosphorylated chain was degraded with spleen a-exonuclease. In this case the w terminus appeared as a nucleoside, the rest of the chain as 3 -mononucleotides. 

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. 

Roblin (74) used venom exonuclease to identify the a terminus in RNA isolated from bacteriophage R-17. The RNA was degraded with alkali and the mixture chromatographed according to Tomlinson and Tener (69). The terminal appeared at the place in the pattern that corre-... 

Venom exonuclease was also used to determine the o> terminus and its adjacent four nucleotides in tobacco mosaic virus (75). The sequence was determined from the rate of appearance of monomers. 

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... 

The mechanism of action of spleen exonuclease is similar to that seen for venom exonuclease (19-21) but different from the processive type of attack exhibited by E. coli RNase II, sheep kidney exonuclease, and polynucleotide phosphorylase (22, 23), in which cases each polynucleotide molecule is completely degraded before the enzymes attack a new molecule. The results of Bernardi and Cantoni (12) contradict the previous beliefs that the enzyme has an intrinsic, though weak, endonucleolytic activity (5) and that a phosphate group in a terminal 5 position makes a polynucleotide chain completely resistant to the enzyme (15, 24, 25). 

Glucosylated oligonucleotides obtained from T4 phage DNA by acid DNase digestion are resistant to spleen exonuclease (28). It has been reported that acetylation of the 2 -OH groups of tRNA completely inhibits the action of the enzyme, whereas venom exonuclease is not affected (29). The naturally occurring methylation of sugars and bases in tRNA does not seem to hinder the action of spleen exonuclease. 

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. 

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