Stereochemical course of DNA hydrolysis by nuclease S1

Nuclease S1 hydrolyzes the Sp-diastereomer of 5'-O-(2'-deoxyadenosyl)-3'-O-thymidyl phosphorothioate in H2(18)O to [18O]deoxyadenosine 5'-O-phosphorothioate which can be phosphorylated enzymatically to the Sp-diastereomer of [alpha-18O]deoxyadenosine 5'-O-(1-thiotriphosphate). 31P nmr spectroscopy shows the oxygen-18 in this compound to be in a nonbridging position at the alpha-phosphorus, indicating that the hydrolysis reaction catalyzed by nuclease S1 proceeds with inversion of configuration at phosphorus. This result is compatible with a direct nucleophilic attack of H2O at phosphorus without the involvement of a covalent enzyme intermediate.     

Stereochemical outcome of the hydrolysis reaction catalyzed by the EcoRV restriction endonuclease.

The stereochemical course of the reaction catalyzed by the EcoRV restriction endonuclease has been determined. This endonuclease recognizes GATATC sequence and cuts between the central T and dA bases. The Rp isomer of d(GACGATsATCGTC) (this dodecamer contains a phosphorothioate rather than the usual phosphate group between the central T and dA residues, indicated by the s) was a substrate for the endonuclease. Performing this reaction in H2 18O gave [18O]dps(ATCGTC) (a pentamer containing an 18O-labeled 5'-phosphorothioate) which was converted to [18O]dAMPS with nuclease P1. This deoxynucleoside 5'-[18O]phosphorothioate was stereospecifically converted to [18O]dATP alpha S with adenylate kinase and pyruvate kinase [Brody, R. S., & Frey, P. A. (1981) Biochemistry 20, 1245-1251]. Analysis of the position of the 18O in this product by 31P NMR spectroscopy showed that it was in a bridging position between the alpha- and beta-phosphorus atoms. This indicates that the EcoRV hydrolysis proceeds with inversion of configuration at phosphorus. The simplest interpretation is that the mechanism of this endonuclease involves a direct in-line attack at phosphorus by H2O with a trigonal bipyramidal transition state. A covalent enzyme oligodeoxynucleotide species can be discounted as an intermediate. An identical result has been previously observed with the EcoR1 endonuclease [Connolly, B. A., Eckstein, F., & Pingoud, A. (1984) J. Biol. Chem. 259, 10760-10763]. X-ray crystallography has shown that both of these endonucleases contain a conserved array of amino acids at their active sites. Possible mechanistic roles for these conserved amino acids in the light of the stereochemical findings are discussed.     

The stereochemical course of the ribulose-5-phosphate kinase-catalyzed reaction

Spinach-leaf ribulose-5-phosphate kinase catalyzes the reaction of (Rp)-[beta, gamma-18O, gamma-18O]adenosine 5'-(3-thiotriphosphate) with ribulose 5-phosphate to form ribulose 1-[18O]phosphorothioate 5-phosphate. This product is incubated with CO2, Mg2+, and ribulose-bisphosphate carboxylase to form the [18O]phosphorothioate of D-glycerate. Reduction of this material using phosphoglycerate kinase/ATP, glyceraldehyde-3-phosphate dehydrogenase/NADH, triose-phosphate isomerase, and glycerol-phosphate dehydrogenase/NADH produces glycerol 3-[18O]phosphorothioate, which is subjected to ring closure using diethylphosphorochloridate. This in-line reaction produces a diastereoisomeric mixture of glycerol 2,3-cyclic phosphorothioates. 31P NMR spectroscopy was used to analyze the 18O content of the products. The anti-diastereoisomer, which is the major isomer formed and corresponds to the downfield 31P NMR signal (Pliura, D.H., Schomburg, D., Richard, J.P., Frey, P.A., and Knowles, J.R. (1980) Biochemistry 19, 325-329), retains the 18O label. This observation indicates that the ribulose-5-phosphate kinase reaction proceeds with inversion of configuration at phosphorus. The reaction is, therefore, unlikely to involve the participation of a covalent phosphoryl-enzyme intermediate.     

Mung bean (Phaseolus aureus) nuclease. A mechanistic investigation of the DNA-cleavage reaction using a dinucleoside phosphorothioate.

Mung-bean (Phaseolus aureus) nuclease has been found to cleave the Sp diastereoisomer of 5'-O-thymidyl 3'-O-(2'-deoxyadenosyl)phosphorothioate, (Sp)-d[Ap(S)T], in 18O-labelled water with inversion of configuration at phosphorus to give (Sp)-thymidine 5'-[16O, 18O]phosphorothioate, the stereochemistry of which was deduced by methylation to (Rp,Sp)-thymidine 5'-S-methyl-O-methyl-[16O,18O]phosphorothioate and 31P-n.m.r. analysis. This result is consistent with a mechanism involving a direct 'in-line' attack of water on DNA for the nuclease-catalysed reaction without the involvement of a covalent nucleotidylated-enzyme intermediate.     

Stereochemical course of the hydrolysis of thymidine 5'-(4-nitrophenyl [17O,18O]phosphate) in H216O catalyzed by the phosphodiesterase from snake venom

The phosphodiesterase from snake venom catalyzes the hydrolysis of the Rp diastereomer of thymidine 5'-(4-nitrophenyl [17O,18O]phosphate) in H216O with retention of configuration at phosphorus. This result is in agreement with those previously reported for the hydrolysis of chiral phosphorothioate substrates (Bryant, F. R., and Benkovic, S. J. (1979) Biochemistry 18, 2825-2828; Burgers, P. M. J., Eckstein, F., and Hunneman, D. H. (1979) J. Biol. Chem. 254, 7476-7478). The hydrolysis reaction catalyzed by this enzyme occurs via formation of a covalent nucleotidylated enzyme intermediate.     

The stereochemical course of hydrolysis catalysed by snake venom 5''-nucleotide phosphodiesterase.

Adenosine 5'-(S)-[16O,17O,18O]phosphate was pyrophosphorylated by the combined action of adenylate kinase and pyruvate kinase. The isotopomers of adenosine 5'-[alpha-16O,17O,18O]triphosphate were hydrolysed by venom 5'-nucleotide phosphodiesterase (Crotalus adamanteus) in H2(17)O. Analysis by 31P nuclear magnetic resonance spectroscopy of the resulting adenosine 5'-[16O,17O,18O]phosphate, after cyclization and esterification, showed that the hydrolysis occurs with retention of configuration at phosphorus. The most likely explanation of this observation is that the enzymic hydrolysis involves a double displacement at phosphorus with a covalent nucleotidyl--enzyme intermediate on the reaction pathway.     

Stereochemistry of hydrolysis of adenosine 3':5'-cyclic phosphorothioate by the cyclic phosphodiesterase from beef heart.

Adenosine 3':5'-cyclic phosphorothioate, Sp-diastereomer was hydrolyzed by cyclic phosphodiesterase from beef heart in the presence of [18O]water to [18O]adenosine 5'-phosphorothioate. This was phosphorylated by myokinase and pyruvate kinase to [18O]adenosine 5'-(1-thiotriphosphate),Sp-diastereomer. The position of 18O was determined to be in a nonbridging position. This result indicates that the hydrolysis proceeded with inversion of configuration at phosphorus.     

Chiral [18O]phosphorothioates. The stereochemical course of thiophosphoryl group transfer catalyzed by nucleoside phosphotransferase.

Nucleoside phosphotransferase from barley seedlings was used to catalyze the equilibration of adenosine-5'-[18O]phosphorothioate having the S configuration at phosphorus with [adenine-8-14C]adenosine to produce [adenine-8-14C]adenosine-5'-[18O]phosphorothioate and adenosine. The configuration of the chiral phosphorus in adenosine-5'-[18O]phosphorothioate which was used as the donor substrate was then compared with that of the [adenine-8-14C]adenosine-5'-[18O]phosphorothioate isolated from the reaction mixture. They were found to be the same, showing that the reaction proceeds with 99.7% retention of configuration of the [18O]phosphorothioate. This is interpreted to be indicative of the involvement of a thiophosphoryl-enzyme intermediate in the nucleoside phosphotransferase reaction. The synthesis of adenosine-5'-[18O]phosphorothioate having the R and S configurations at the phosphorus atoms is described.     

The stereochemical course of the reaction catalyzed by soluble bovine lung guanylate cyclase

The stereochemical course of the reaction catalyzed by the soluble form of bovine lung guanylate cyclase has been investigated using [alpha-18O]guanosine 5'-triphosphate (Rp diastereomer) and guanosine 5'-O-(1-thiotriphosphate) (Sp diastereomer) as substrates. The product from the 3-thiomorpholino-1',1'-dioxide sydnonimine-stimulated enzymatic cyclization of [alpha-18O] guanosine 5'-triphosphate was esterified with diazomethane. 31P NMR analysis of the triesters indicated that all of the 18O label was present in the axial position. Guanosine 5'-O-(1-thiotriphosphate) (Sp diastereomer) was cyclized under stimulated and basal enzyme activities and, in both cases, the Rp diastereomer of guanosine 3',5'-cyclic phosphorothioate was formed. This was determined by direct comparison with material synthesized chemically from guanosine 5'-phosphorothioate. The results from these experiments show that the reaction catalyzed by guanylate cyclase proceeds with inversion of configuration at phosphorus and this indicates that the reaction proceeds by way of a single direct displacement reaction.     

Streospecific substitution of oxygen-18 for sulfur in nucleoside phosphorothioates

Reaction of nucleoside phosphorothioates with N-bromosuccinimide in dioxane and H218O leads to the exchange of sulfur for oxygen-18. Using the Sp-isomers of adenosine 5'-O-(1-thiodiphosphate) and adenosine 3',5'-cyclic phosphorothioate, it can be shown by 31P NMR spectroscopy that this reaction proceeds with inversion of configuration yielding the Rp-isomers of [alpha-18O]ADP and [18O]cAMP, respectively. Adenosine 5'-O-(2-thiotriphosphate) and adenosine 5'-O-(3-thiotriphosphate) are likewise converted to [beta-18O]ATP and [gamma-18O]ATP although the stereochemistry of the former reaction has yet to be evaluated. With very slight modifications this reaction is applicable to all the common bases.     

Stereochemical course of the 3'----5'-exonuclease activity of DNA polymerase I

(Sp)-2'-Deoxyadenosine 5'-O-[1-17O,1-18O,1,2-18O]triphosphate has been synthesized by desulfurization of (Sp)-2'-deoxyadenosine 5'-O-(1-thio[1,1-18O2]diphosphate) with N-bromosuccinimide in [17O]water, followed by phosphorylation with phosphoenolpyruvate-pyruvate kinase. A careful characterization of the product using high-resolution 31P NMR revealed that the desulfurization reaction proceeded with approximately 88% direct in-line attack at the alpha-phosphorus and 12% participation by the beta-phosphate to form a cyclic alpha,beta-diphosphate. The latter intermediate underwent hydrolysis by a predominant nucleophilic attack on the beta-phosphate. This complexity of the desulfurization reaction, however, does not affect the stereochemical integrity of the product but rather causes a minor dilution with nonchiral species. The usefulness of the (Sp)-2'-deoxyadenosine 5'-O-[1-17O,1-18O,1,2-18O]triphosphate in determining the stereochemical course of deoxyribonucleotidyl-transfer enzymes is demonstrated by using it to delineate the stereochemical course of the 3'----5'-exonuclease activity of DNA polymerase I. Upon incubation of this oxygen-chiral substrate with Klenow fragment of DNA polymerase I in the presence of poly[d(A-T)] and Mg2+, a quantitative conversion into 2'-deoxyadenosine 5'-O-[16O,17O,18O]monophosphate was observed. The stereochemistry of this product was determined to be Rp. Since the overall template-primer-dependent conversion of a deoxynucleoside triphosphate into the deoxynucleoside monophosphate involves incorporation into the polymer followed by excision by the 3'----5'-exonuclease activity and since the stereochemical course of the incorporation reaction is known to be inversion, it can be concluded that the stereochemical course of the 3'----5'-exonuclease is also inversion.     

Synthesis and characterization of an octanucleotide containing the EcoRI recognition sequence with a phosphorothioate group at the cleavage site

The synthesis and characterization of an octanucleotide, d(GGsAATTCC), containing the recognition sequence of the EcoRI restriction endonuclease with a phosphorothioate internucleotidic linkage at the cleavage site are described. Two approaches for the synthesis of the RP and SP diastereomers of this octamer by the phosphite method are presented. The first consists of the addition of sulfur instead of H2O to the phosphite at the appropriate position during chain elongation. This method results in a mixture of diastereomers that can be separated by high-performance liquid chromatography after 5'-terminal phosphorylation. The second uses the presynthesized and diastereomerically pure dinucleoside phosphorothioate d[Gp(S)A] for the addition to the growing oligonucleotide chain as a block. The products are characterized by digestion with nuclease P1, fast atom bombardment mass spectrometry, 31P NMR spectroscopy, and conversion to d(GGAATTCC) by desulfurization with iodine. Only the RP diastereomers of d(GGsAATTCC) and its 5'-phosphorylated derivative are cleaved by EcoRI endonuclease. The rate of hydrolysis is slower than that of the unmodified octamer. The phosphorothioate octamer will be useful for the determination of the stereochemical course of the EcoRI-catalyzed reaction.     

Gentamicin nucleotidyltransferase. Stereochemical inversion at phosphorus in enzymatic 2'-deoxyadenylyl transfer to tobramycin

Gentamicin nucleotidyltransferase-catalyzed reaction of (Sp)-[alpha-17O]dATP with tobramycin produced 2"-(2'-deoxyadenosine 5'-[17O]phosphoryl)tobramycin. The configuration at phosphorus in this product was shown to be Rp by chemical degradation to chiral [17O, 18O]dAMP using a stereochemically defined procedure, and determination of the configuration at phosphorus in this product. Periodate-base treatment of 2"-(2'-deoxyadenosine 5'-[17O]phosphoryl)tobramycin followed by NaBH4 reduction produced (2-glyceryl)-[17O]dAMP, which upon snake venom phosphodiesterase-catalyzed hydrolysis in H(2)18O produced [17O,18O] dAMP. The configuration at phosphorus in this product was shown to be S by enzymatic phosphorylation to [17O,18O]dATP, adenylylcyclase (Bordetella pertussis)-catalyzed cyclization to 3',5'-cyclic [17O,18O]dAMP, and 31P NMR analysis of the ethyl esters. Since snake venom phosphodiesterase-catalyzed hydrolyses proceed with retention of configuration at phosphorus, (Sp)-[17O,18O]dAMP must have been produced from (Rp)-(2-glyceryl)-[17O]dAMP; and since the chemical degradation to the latter compound did not involve cleavage of any bonds to phosphorus, the initial enzymatic product must have been (Rp)-2"-(2'-deoxyadenosine 5'-[17O]phosphoryl)tobramycin. Therefore, nucleotidyl transfer catalyzed by gentamicin nucleotidyl-transferase proceeds with inversion of configuration at phosphorus, and the reaction mechanism involves an uneven number of phosphotransfer steps. Inasmuch as this is an uncomplicated two-substrate group transfer reaction, the mechanism probably involves direct nucleotidyl transfer from the nucleoside triphosphate to the aminoglycoside. The B. pertussis adenylylcyclase reaction was shown to proceed with inversion at phosphorus, as has been established for other adenylylcyclases.     

Stereochemical course of the reaction catalyzed by the cyclic GMP phosphodiesterase from retinal rod outer segments

The stereochemical course of hydrolysis catalyzed by the cyclic GMP phosphodiesterase from bovine retinal rod outer segments was determined. The Sp diastereomer of guanosine 3',5'-cyclic monophosphorothioate was hydrolyzed by cyclic GMP phosphodiesterase in H2(18)O to give [16O,18O]guanosine 5'-monophosphorothioate. This isotopomer was reacted with diphenyl phosphorochloridate to form the two diastereomers of P1-(5'-guanosyl) P2-(diphenyl) 1-thiodiphosphate. The 31P NMR spectrum of this mixture of diastereomers was identical to that obtained from [16O,18O]guanosine 5'-monophosphorothioate resulting from the hydrolysis of the Rp diastereomer of guanosine 5'-p-nitrophenyl phosphorothioate by snake venom phosphodiesterase. This finding indicates that the 18O is bridging in the Rp diastereomer of the P1-(5'-guanosyl) P2-(diphenyl) 1-thiodiphosphate and nonbridging in the Sp diastereomer. As the snake venom phosphodiesterase reaction is known to proceed with retention of configuration, it follows that hydrolysis by retinal rod cyclic GMP phosphodiesterase proceeds with inversion of configuration at the phosphorus atom.     

Unambiguous stereochemical course of rabbit liver fructose bisphosphatase hydrolysis

The stereochemical course of rabbit liver fructose bisphosphatase (EC 3.1.3.11) was determined by hydrolyzing the substrate analogue (Sp)-[1-18O]fructose 1-phosphorothioate 6-phosphate in H(2)17O, incorporating the chiral, inorganic phosphorothioate product into adenosine 5'-O-(2-thiotriphosphate) (ATP beta S), and analyzing the isotopic distribution of 18O in ATP beta S by 31P NMR. The result indicates that the 1-phosphoryl group is transferred with inversion of configuration. A series of single-turnover experiments ruled out an acyl phosphate intermediate in the hydrolysis. Consequently, fructose bisphosphatase catalyzes the hydrolysis of fructose 1,6-bisphosphate via a direct transfer of the phosphoryl moiety to water.     

A new method for determining the stereochemistry of DNA cleavage reactions: application to the SfiI and HpaII restriction endonucleases and to the MuA transposase

A new method was developed for tracking the stereochemical path of enzymatic cleavage of DNA. DNA with a phosphorothioate of known chirality at the scissile bond is cleaved by the enzyme in H218O. The cleavage produces a DNA molecule with the 5'-[16O,18O, S]-thiophosphoryl group, whose chirality depends on whether the cleavage reaction proceeds by a single-step hydrolysis mechanism or by a two-step mechanism involving a protein-DNA covalent intermediate. To determine this chirality, the cleaved DNA is joined to an oligonucleotide by DNA ligase. Given the strict stereochemistry of the DNA ligase reaction, determined here, the original chirality of the phosphorothioate dictates whether the 18O is retained or lost in the ligation product, which can be determined by mass spectrometry. This method has advantages over previous methods in that it is not restricted to particular DNA sequences, requires substantially less material, and avoids purification of the products at intermediate stages in the procedure. The method was validated by confirming that DNA cleavage by the EcoRI restriction endonuclease causes inversion of configuration at the scissile phosphate. It was then applied to the reactions of the SfiI and HpaII endonucleases and the MuA transposase. In all three cases, DNA cleavage proceeded with inversion of configuration, indicating direct hydrolysis of the phosphodiester bond by water as opposed to a reaction involving a covalent enzyme-DNA intermediate.     

Stereochemistry of the hydrolysis of adenosine 5'-thiophosphate catalyzed by venom 5'-nucleotidase

The stereochemical problem involving a pro-pro-prochiral phosphorus center, the hydrolysis of adenosine 5'-monophosphate to adenosine and inorganic phosphate catalyzed by the venom 5'-nucleotidase, has been studied by use of chiral [16O, 17O, 18O]thiophosphates (Psi). (Rp)- and (Sp)-[alpha-18O1]Adenosine 5'-thiophosphates (AMPS) were synthesized by a combined chemical and biochemical procedure. Hydrolysis of (Rp)- and (Sp)-[alpha-18O1]AMPS in H217O by 5'-nucleotidase gave two enantiomers of chiral Psi of unknown configuration. A 31P NMR method based on the combination of the quadrupolar effect of 17O [Tsai, M.-D. (1979) Biochemistry 18, 1468-1472] and the 18O isotope shift [Cohn, M., & Hu. A. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 200-203] has been developed to analyze the configuration of chiral Pso. The results indicate that hydrolysis of (Rp)- and (Sp)-[alpha-18O1]AMPS in H217O gave (R)- and (S)- [16O, 17O, 18O]Psi, respectively. Therefore the hydrolysis of AMPS catalyze by the venom 5'-nucleotidase must proceed with inversion of configuration at phosphorus, which suggests that the reaction is most likely an "in line" single displacement without involving a phosphoryl-enzyme intermediate and without pseudorotation.     

The stereochemical course of thiophosphoryl group transfer catalyzed by T4 polynucleotide kinase

The stereochemical course of the phosphoryl transfer reaction catalyzed by T4 polynucleotide kinase has been determined using the chiral ATP analog, (Sp)-adenosine-5'-(3-thio-3-[18O]triphosphate). T4 polynucleotide kinase catalyzes the transfer of the gamma-thiophosphoryl group of (Sp)-adenosine-5'-(3-thio-3-[18O]triphosphate) to the 5'-hydroxyl group of ApA to give the thiophosphorylated dinucleotide adenyl-5'-[18O]phosphorothioate-(3'-5')adenosine. A sample of adenyl-5'-[18O]phosphorothioate-(3'-5')adenosine was subjected to venom phosphodiesterase digestion. The resulting adenosine-5'-[18O]phosphorothioate was shown to have the Rp configuration, thus indicating that the thiophosphoryl transfer reaction occurs with overall inversion of configuration of phosphorus.     

The mechanism of guanosine nucleotide hydrolysis by p21 c-Ha-ras. The stereochemical course of the GTPase reaction

The use of guanosine 5'-O-(gamma-thio)triphosphate as a substrate for p21 c-Ha-ras was established. By using chirally labeled [gamma-17O,18O]guanosine 5'-O-(gamma-thio)triphosphate, the stereochemical course of the GTPase reaction was determined. The analysis shows that the hydrolysis occurs with inversion at the gamma-phosphorus. This shows that the most likely mechanism is a single step, in-line transfer, without a phosphoenzyme or other phosphorylated intermediate.     

Stereochemical retention of the configuration in the action of Fhit on phosphorus-chiral substrates

Fhit is the protein product of FHIT, a candidate human tumor suppressor gene. Fhit catalyzes the hydrolysis of diadenosine triphosphate (Ap3A) to AMP and ADP. Fhit is here shown to catalyze the hydrolysis in H218O with production of adenosine 5'-[18O]phosphate and ADP, proving that the substitution of water is at Palpha and not at Pbeta. The chain fold of Fhit is similar to that of galactose-1-phosphate uridylyltransferase, which functions by a double-displacement mechanism through the formation of a covalent nucleotidyl-enzyme intermediate and overall retention of configuration at Palpha. The active site of Fhit contains a histidine motif that is reminiscent of the HPH motif in galactose-1-phosphate uridylyltransferases, in which the first histidine residue serves as the nucleophilic catalyst to which the nucleotidyl group is bonded covalently in the covalent intermediate. In this work, the Fhit-catalyzed cleavage of (RP)- and (SP)-gamma-(m-nitrobenzyl) adenosine 5'-O-1-thiotriphosphate (mNBATPalphaS) in H218O to adenosine 5'-[18O]thiophosphate is shown to proceed with overall retention of configuration at phosphorus. gamma-(m-Nitrobenzyl) adenosine 5'-O-triphosphate (mNBATP) is approximately as good a substrate for Fhit as Ap3A, and both (RP)- and (SP)-mNBATPalphaS are substrates that react at about 0.5% of the rate of Ap3A. The stereochemical evidence indicates that hydrolysis by Fhit proceeds by a double-displacement mechanism, presumably through a covalent AMP-enzyme intermediate.