Stereochemical course of the reaction catalyzed by 5'-nucleotide phosphodiesterase from snake venom

Bryant, Floyd R.; Benkovic, Stephen J.

doi:10.1021/bi00580a022

Cited by 145 publications

(47 citation statements)

References 21 publications

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“…3 The results of Hae III digestion of intact pBR322, in parallel with those of the shortened plasmid, are shown in Fig. 4 Right, lanes 1 (10,20), kinases (21), exonucleases (11,22), and of nucleotidyltransferases (23) have been explored with these compounds. Their significance for such studies is that the stereochemistry of the reaction about the chiral phosphorus atom can easily be determined.…”

Section: Resultsmentioning

confidence: 99%

A DNA fragment with an alpha-phosphorothioate nucleotide at one end is asymmetrically blocked from digestion by exonuclease III and can be replicated in vivo.

Putney¹,

Benkovic²,

Schimmel³

1981

Proc. Natl. Acad. Sci. U.S.A.

180

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2'-Deoxyadenosine 5'-O-(1-thiotriphosphate) (dATP [caS]) was introduced into the 3' ends of DNA restriction fragments with Escherichia coli DNA polymerase I to give phosphorothioate internucleotide linkages. Such "capped" 3' ends were found to. be resistant to exonuclease III digestion. Moreover, the resistance to digestion is great enough that, under conditions used by us, just one strand ofa double helix is digested by exonuclease III when a cap is placed at only one end; when digestion is carried to completion, this results in production of intact single strands. When digestion with exonuclease mI is limited and is followed by SI nuclease treatment, double-stranded DNA fragments asymmetrically shortened from just one side are produced. In this way thousands of nucleotides can be selectively removed from one end of a restriction fragment. In vitro introduction of phosphorothioate linkages into one end of a linearized replicative plasmid, followed by exonuclease III and SI nuclease treatments, gives rise to truncated forms that, upon circularization by blunt-end ligation, transform E. coli and replicate in vivo.It is often useful to decrease the length ofa given DNA fragment in a progressive, controlled manner (1-7). Two different schemes are currently employed for progressively varying the length of double-stranded DNA fragments. One is the use of exonuclease III followed by single-strand-specific S1 nuclease (1,2,4,5,7,8). Another scheme uses Bal 31 nuclease (3, 6), which simultaneously digests both 5' and 3' ends of a doublestranded DNA fragment (9). The primary disadvantage of these approaches is that the nucleases act simultaneously at both ends of the fragment so that desirable sequences at one end are not preserved.The Sp diastereomer of the nucleoside a-thiotriphosphates (which have a sulfur substituted for an oxygen at the a-phosphate) act as substrates for Escherichia coli DNA polymerase I in DNA synthesis (10). These compounds, although incorporated at an efficiency similar to that of the natural substrates, effectively block the 3' exonuclease activity of DNA polymerase I at the phosphorothioate nucleotide linkage (unpublished results). In work reported here we found that placement of an athionucleotide at only one of the 3' ends of a DNA fragment effectively blocks that end from digestion with exonuclease III. Digestion proceeds progressively from the opposite end and, after S1 nuclease treatment, the fragment is shortened with the integrity of the nucleotide sequence at the "capped" end preserved. Alternatively, if exonuclease III digestion is allowed to proceed to completion, a full-length single strand is generated from the original double strand.This work also demonstrates that, when 2'-deoxyadenosine 5'-O-(l-thiotriphosphate) (dATP [aS]) is inserted into 3' recessed ends ofrestriction fragments, the phosphorothioate linkage allows subsequent ligation ofthe DNA, restriction ofligated junctions, and in vivo replication of plasmids containing phosphorothioate nucleotide linkages. Purific...

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Section: Resultsmentioning

confidence: 99%

A DNA fragment with an alpha-phosphorothioate nucleotide at one end is asymmetrically blocked from digestion by exonuclease III and can be replicated in vivo.

Putney¹,

Benkovic²,

Schimmel³

1981

Proc. Natl. Acad. Sci. U.S.A.

180

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“…Thus, the inversion incurred during E-P formation should be reversed by a second inversion as E-P reacts with acceptor, the two inversions resulting in net retention. And, indeed, such net retentions have been found for galactose-l-P uridylyltransferase (EC 2.7.7.10) (17), the classical NDP kinase (17), alkaline phosphatase (EC 3.1.3.1) (18), and phosphodiesterase I (EC 3.1.4.1) (19,20), all of which have a double-displacement mechanism with a demonstrated E-P on the reaction pathway. * But acetate kinase with an E-P on its pathway catalyzes not a retention but a net inversion on phosphorus.…”

Section: Review Of Available Datamentioning

confidence: 91%

Acetate kinase: a triple-displacement enzyme.

Spector¹

1980

Proc. Natl. Acad. Sci. U.S.A.

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Facts relating to the mechanism of phosphoryl transfer by acetate kinase (ATP:acetate phosphotransferase, EC 2.7.2.1) are reviewed. They point to the existence of at least one experimentally established phosphoenzyme (E-P) intermediate on the reaction pathway. Sterically, the phosphoryl transfer occurs with a net inversion of the configuration of the phosphorus atom. These facts are best in accord with a triple-displacement mode of action for acetate kinase, with two E-P intermediates and three steric inversions on phosphorus. It follows that a second E-P for acetate kinase must exist.Ever since its discovery by Lipmann (1), acetate kinase (EC 2.7.2.1) has engaged the active interest of students of enzyme mechanism, and never more so than now. The enzyme catalyzes the synthesis of ATP from acetyl-P. In the reverse direction the enzyme is the prototypic carboxylate kinase. As such, the chemical means by which acetate kinase effects phosphoryl transfer has been much studied, with broad implications in the wider field of phosphotransferases generally.REVIEW OF AVAILABLE DATA The equilibrium constant of reaction 1, as catalyzed by the Escherichia coli enzyme, is reported to range from 200 to 1500 (1-5), indicating that, thermodynamically, the reaction greatly favors ATP synthesis from acetyl-P. In its specificity, the enzyme is most active on acetyl-P but has some activity on propionyl-P (30%) and carbamoyl-P (18%), and none on butyryl-P, glutaryl-P, and phosphoenolpyruvate (4). The purines GTP, ITP, and dATP, as well as ATP, are excellent substrates (2, 4), accounting thus for the intrinsic purine nucleoside-5'-diphosphate kinase (NDP kinase, EC 2.7.4.6) activity of acetate kinase (6, 7).It is curious that the Km for acetate is 300 mM (2), one of the highest Michaelis constants on record. Another curiosity is the cold inactivation to which acetate kinase is liable (8). Chilled at 0°C, the enzyme quickly loses activity. Restoration of activity is possible; it is most rapidly (2-3 min) achieved, even at 0°C, by the addition of nucleotide (4). Acetate and acetyl-P are without effect. Reactivation in the absence of nucleotide requires a long-term (1-2 hr) incubation at room temperature in the presence of Mg2+ and a monovalent cation (7,8). If these inactivation-reactivation phenomena are due to conformational changes in the protein, as seems probable, then it is clear that only the nucleotides, of the four substrates of reaction 1, can specifically induce the active conformation of the enzyme.Phosphorylation of Acetate Kinase. Fig. 1 shows that the enzyme can be phosphorylated with ATP or acetyl-P, and the phosphoenzyme (E-P) can be isolated by gel filtration (6, 9, 10). The E-P prepared with ATP and the E-P prepared with acetyl-P are identical with respect to pH-stability, sensitivity to hydroxylamine, and the capacity to phosphorylate ADP or acetate (4). The phosphoryl group in E-P is linked to a carboxyl of the enzyme (9). Reduction of E-P with boro[3H]hydride (and isolation, after hydrolysis, of tritiated a...

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“…When rate data were transformed to the alcohol efficiency parameter EA [see Eqn (1) in the Introduction], it became apparent that the primary alcohol functional groups of glycerol phosphates were much better acceptors of the SVP-bound AMP than water (Fig. 5).…”

Section: Efficiency Of Glycerol Phosphates Asmentioning

confidence: 99%

High Efficiency of Glycerol 2‐Phosphate and sn ‐Glycerol 3‐Phosphate as Nucleotidyl Acceptors in Snake Venom Phosphodiesterase Esterifications

Vergeles

García-Díaz

Cameselle

1995

European Journal of Biochemistry

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Snake venom phosphodiesterase (SVP) catalyzes the alcoholysis of ATP by primary R-CH,OH alcohols with uncharged R residues, yielding AMP-0-CH,R esterification products. The alcohols compete with water for an SVP-bound adenylyl intermediate. In this study, it has been shown that SVP also catalyzes the reactions of glycerol 2-phosphate and sn-glycerol 3-phosphate with ATP to yield AMP-Oglycerophosphoryl esters. The products were identified by HPLC, the dependency of the reactions on glycerol phosphates, ultraviolet spectroscopy, and conversion to AMP by phosphodiesterase, or to AMP-0-glyceryl esters by alkaline phosphatase. The results demonstrated that R-CH,OH alcohols with negatively charged R residues, as well as secondary alcohols, act as adenylyl acceptors in SVP reactions, thus extending the usefulness of SVP as a tool to produce 5'-nucleotide derivatives. The efficiencies (E,) of glycerol phosphates as adenylyl acceptors were very high at low, millimolar concentrations, but decreased abruptly when the acceptor concentration was increased and, for glycerol 2-phosphate, when P, or NaCl was present. In contrast, glycerol E, was independent of its own concentration, P,, and NaCI. The responses of glycerol phosphates indicate that they act as adenylyl acceptors via a mechanism different from uncharged R-CH,OH alcohols. The occurrence of an acceptor-binding enzyme site, specific for negatively charged R residues, and its potential relevance to the in vivo role of 5'-nucleotide phosphodiesterases as 5'-nucleotidyl transferases are discussed.Keyvvords: phosphodiesterase-I ; nucleotide-pyrophosphatase ; esterification ; 5'-nucleotide esters ; glycerol phosphates.Snake venom 5'-nucleotide phosphodiesterase (SVP) catalyzes the solvolysis of phosphodiester and phosphoanhydride derivatives of 5'-nucleotides. The reaction sequence includes a double displacement, with the formation of a 5'-nucleotidyl intermediate covalently bound to the enzyme 11-51 and its transfer to an acceptor that can be water or an R-CH,OH alcohol [6, 71. The solvolysis of ATP in water/alcohol mixtures follows a branched route (Scheme 1) 171.Reaction rates with different acceptors may reflect both the reactivities of the alcohols and their influence on enzyme structure and activity. Both of these properties depend on the nature and concentration of the alcohol but are unrelated to each other. Therefore, to compare acceptor reactivities without interference by enzyme (in)activation effects, the parameter EA (alcohol acceptor efficiency) is very convenient (71 :For an alcohol as reactive as water, E, = 1. since in this case the mole fraction of the esterification product AMP-0-CH,R equals the mole fraction of alcohol as an acceptor.Comparisons of E, values disclose valuable mechanistic information. Short-chain R-CH,OH alcohols with uncharged R residues, display E,% values that are largely concentration independent, but depend on the nature of R. For alcohols with a hydroxyl group pK., 3 15.5, E, relates to electronic factors like the acid strength: t...

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Stereochemical course of the reaction catalyzed by 5'-nucleotide phosphodiesterase from snake venom

Cited by 145 publications

References 21 publications

A DNA fragment with an alpha-phosphorothioate nucleotide at one end is asymmetrically blocked from digestion by exonuclease III and can be replicated in vivo.

A DNA fragment with an alpha-phosphorothioate nucleotide at one end is asymmetrically blocked from digestion by exonuclease III and can be replicated in vivo.

Acetate kinase: a triple-displacement enzyme.

High Efficiency of Glycerol 2‐Phosphate and sn ‐Glycerol 3‐Phosphate as Nucleotidyl Acceptors in Snake Venom Phosphodiesterase Esterifications

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