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.     

Stereochemistry and mechanism of the GDP-mannose dehydratase reaction.

The reaction catalyzed by bacterial GDP-mannose dehydratase (E.C. 4.2.1.47), the conversion of GDP-D-mannose to GDP-4-keto-6-deoxymannose (GDP-6-deoxy-D-lyxo-hexos-4-ulose), was studied with (6R)- and (6S)-GDP-D-[4-2H1,6-3H]mannose. Conversion of these stereospecifically labeled substrates in the presence of excess unlabeled GDP-mannose into the 4-keto-6-deoxy derivatives followed by Kuhn-Roth oxidation gave acetic acid samples which were subjected to configurational analysis of the isotopically chiral methyl group. The observed F values of 64 for the material from the (6S) substrate and 31 for that from the (6R) isomer, corresponding to 48% e.e. R and 66% e.e. S configuration, respectively, of the methyl group indicate that (a) the oxidoreductase reaction involves transfer of H-4 to C-6, (b) the transfer is predominantly intramolecular, and (c) the transfer is stereospecific, H-4 replacing the C-6 hydroxyl group with inversion of configuration. A mechanism for the reaction is proposed on the basis of these results.     

Stereochemistry of phosphoryl transfer

A general method has been developed for the synthesis of chiral [16O,17O,18O]phosphate monoesters of known absolute configuration. An analytic method for determining the absolute configuration of chiral phosphate esters has also been developed, which is based on the isotope effects of 17O and 18O at phosphorus in the 31P nuclear magnetic resonance spectrum. These methods have shown that phosphoryl transfer catalysed by hexokinase, phosphofructokinase and pyruvate kinase occurs with inversion of configuration. This is most simply interpreted as an "in-line' transfer of the phosphoryl group between substrates in the enzyme-substrate ternary complex.     

Phospholipids chiral at phosphorus. Synthesis of chiral phosphatidylcholine and stereochemistry of phospholipase D

Chirally labeled 1,2-dipalmitoyl-sn-glycero-3-phosphocholines (DPPC) with known configuration were synthesized by N-methylation of chirally labeled 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE). Transphosphatidylation of (RP)- and (SP)-[18O]DPPC catalyzed by phospholipase D from cabbage gave (RP)- and (SP)-[18O]DPPE, respectively, as indicated by 31P nuclear magnetic resonance (NMR) analysis of [18O]DPPE. Therefore, phospholipase D catalyzes transphosphatidylation with overall retention of configuration at phosphorus. The steric course of hydrolysis of DPPC catalyzed by the same enzyme was elucidated by the following procedures. Hydrolysis of (RP)-[17O, 18O]DPPC by phospholipase D gave 1,2-dipalmitoyl-sn-glycero-3-[ 16O , 17O, 18O]phosphate ( [ 16O , 17O, 18O] DPPA ) with unknown configuration. The latter compound was then converted to 1-[ 16O , 17O, 18O]phospho-(R)-propane-1,2-diol by a procedure involving no P-O bond cleavage [ Bruzik , K., & Tsai, M.-D. (1984) J. Am. Chem. Soc. 106, 747-754]. The configuration of the phosphopropane -1,2-diol was determined as RP by 31P NMR analysis following ring closure and methylation [ Buchwald , S. L., & Knowles, J. R. (1980) J. Am. Chem. Soc. 102, 6601-6603]. The results indicated that hydrolysis of DPPC catalyzed by phospholipase D also proceeds with retention of configuration at phosphorus. Our results therefore support a two-step mechanism involving a phosphatidyl-enzyme intermediate in the reactions catalyzed by phospholipase D from cabbage.     

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 stereochemical course of yeast hexokinase-catalysed phosphoryl transfer by using adenosine 5''[gamma(S)-16O,17O,18O]triphosphate as substrate.

Adenosine 5'[gamma(S)-16O, 17O, 18O]triphosphate has been synthesized and used to determine the stereochemical course of phosphoryl transfer catalysed by yeast hexokinase. The chirality at phosphorus of the D-glucose 6-[16O,17O,18O]phosphate formed was analysed, after cyclization and methylation, by 31P n.m.r. spectroscopy. The phosphoryl transfer was found to occur with inversion of configuration, with a stereoselectivity in excess of 94%. The simplest interpretation of this result is that the phosphoryl group is transferred between substrates in the enzyme-substrate ternary complex by an 'in line' mechanism.     

Structure of the metal-nucleotide complex in the acetate kinase reaction. A study with gamma-32P-labeled phosphorothioate analogs of ATP

The synthesis of the gamma-32P-labeled diastereomers of adenosine 5'-O-(1-thiotriphosphate) (ATP alpha S) and the Sp isomer of adenosine 5'-O-(2-thiotriphosphate) (ATP beta S) by a modification of the Glynn and Chappell method (Glynn, I. M., and Chappell, J. T., (1964) Biochem. J. 90, 147-149) is described. These analogs were tested as substrates for acetate kinase in the presence of several divalent metal ions. Both isomers of ATP alpha S are substrates in the presence of Mg2+, Mn2+, Co2+, Zn2+, and Cd2+, the Sp isomer being preferred by a factor of between 4.8 (Mg2+) and 52.5 (Cd2+). Only the Rp isomer of ATP beta S is a substrate in the presence of Mg2+, and the Sp isomer becomes a better substrate in the presence of Mn2+, Co2+, and Zn2+; both isomers are equally good substrates in the presence of Cd2+. The change in specificity upon replacing Mg2+ by Cd2+ is greater than 1800 at beta-phosphorus and 10 at alpha phosphorus. These results provide a basis for proposing that the lambda screw sense configuration of the beta, gamma-bidentate MgATP complex is the substrate for acetate kinase. In the reverse reaction, both Sp and Rp isomers of ADP alpha S are substrates in the presence of all metal ions tested, the Sp isomer preferred by a factor between 12.3 (Mg2+) and 45.5 (Cd2+). In the presence of Mg2+, Mn2+, and Co2+, only the Rp isomer of ATP beta S is synthesized from prochiral ADP beta S, while a mixture of Rp and Sp isomers is synthesized in the presence of Zn2+ and Cd2+. These results are analogous to those for the forward reaction and suggest that the Mg.ADP complex which binds as a substrate in the reverse reaction, and is released as a product in the forward reaction, is the beta-monodentate. The classification of acetate kinase as an enzyme having a type I mechanism (Dunaway-Mariano, D. and Cleland, W. W. (1980) Biochemistry 19, 1506-1515) for kinases, is discussed.     

Fluorine NMR studies on stereochemical aspects of reactions catalyzed by transcarboxylase, pyruvate kinase, and enzyme I

The stereochemistry of the transcarboxylase-catalyzed carboxylation of 3-fluoropyruvate has been studied by using fluorine NMR of unpurified reaction mixtures. When the product 3-fluorooxaloacetate was trapped by using malate dehydrogenase, only the 2R,3R diastereomer of 3-fluoromalate was formed. The fluoromethyl group of fluoropyruvate does not take up deuterium label from the solvent during the reaction. These results confirm and extend those obtained previously by Walsh and co-workers [Goldstein, J. A., Cheung, Y. F., Marletta, M. A., & Walsh, C. (1978) Biochemistry 17, 5567-5575] showing that transcarboxylase is specific for one of the two prochiral hydrogens in fluoropyruvate. Transcarboxylase, coupled to malate dehydrogenase, has been used to analyze samples of chiral fluoropyruvate obtained by dephosphorylation of (Z)-fluorophosphoenolpyruvate in D2O in the presence of either pyruvate kinase or enzyme I from the Escherichia coli sugar transport systems. Analysis of the fluoromalate produced showed that fluoroenolpyruvate is deuterated from opposite faces by these two enzymes: enzyme I protonates (deuterates) fluoroenolpyruvate exclusively from the 2-re face and pyruvate kinase does so mainly from the 2-si face. Fluoropyruvate is carboxylated by transcarboxylase with absolute retention of configuration.     

The stereochemical course of phosphoryl transfer catalyzed by herpes simplex virus type I-induced thymidine kinase

Herpes simplex virus type I (HSV-I)-induced thymidine kinase has been shown to catalyze phosphoryl transfer from adenosine 5'-[gamma-(S)-16O,17O,18O]triphosphate to thymidine with inversion of configuration at phosphorus. The simplest interpretation of this result is that phosphoryl transfer occurs by a single in-line group transfer between ATP and thymidine within the ternary enzyme complex.     

Acetate kinase from Veillonella alcalescens. Purification and physical properties.

Acetate kinase (ATP:phosphotransferase E.C.2.7.2.1) has been purified to a high state of purity from Veillonella alcalescens. The native enzyme had a molecular weight of 88,000, as determined by Sephadex G-150 gel filtration. The molecular weight of the monomeric enzyme, estimated from sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was 42,000. The enzyme was determined to be a homodimer from the amino acid composition and the results of trypsin digestion and cyanogen bromide cleavage. Two moles of phosphate were incorporated into the dimer upon incubation of the enzyme with ATP and acetate. These results support the conclusion that each subunit of the dimeric enzyme consists of a single active catalytic center. Succinate enhanced the rate of ATP-ADP phosphoryl group exchange 20-fold and the binding of ATP 10-fold. These results are considered in light of data from previous reports (Pelroy, R. A., and Whiteley, H. R. (1971) J. Bacteriol. 105, 259-267; Bowman, C. M., Valdez, R. O., and Nishimura, J. S. (1976) J. Biol. Chem 251, 3117-3121).     

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.     

Modulation of yeast Sln1 kinase activity by the CCW12 cell wall protein

The yeast Sln1p sensor kinase is best known as an osmosensor involved in the regulation of the hyperosmolarity glycerol mitogen-activated protein kinase cascade. Down-regulation of Sln1 kinase activity occurs under hypertonic conditions and leads to phosphorylation of the Hog1p mitogen-activated protein kinase and increased osmotic stress-response gene expression. Conditions leading to kinase up-regulation include osmotic imbalance caused by glycerol retention in the glycerol channel mutant, fps1 (Tao, W., Deschenes, R. J., and Fassler, J. S. (1999) J. Biol. Chem. 274, 360-367). The hypothesis that Sln1p kinase activity is responsive to turgor was first suggested by the increased Sln1p kinase activity in mutants lacking Fps1p in which glycerol accumulation leads to water uptake. Also consistent with the turgor hypothesis is the observation that reduced turgor caused by treatment of cells with nystatin, a drug that increases membrane permeability and causes cell shrinkage, reduced Sln1p kinase activity (Tao, W., Deschenes, R. J., and Fassler, J. S. (1999) J. Biol. Chem. 274, 360-367; Reiser, V., Raitt, D. C., and Saito, H. (2003) J. Cell Biol. 161, 1035-1040). The turgor hypothesis is revisited here in the context of the identification and characterization of the cell wall gene, CCW12, as a determinant of Sln1p activity. Results of this analysis suggest that the activity of the plasma membrane localized Sln1p is affected by the presence or absence of specific outer cell wall proteins and that this effect is independent of turgor.     

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.     

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.     

Synthesis of stereospecifically deuterated phenylalamines and determination of their configuration.

1. Starting from trans-cinnamic acid a chiral (-)3-phenyl-[2,3-2H]propionic acid has been synthesized using Clostridium kiuyveri cells as catalyst. 2. The chiral dideuterated acid has been converted by chemical methods to a mixture of (2R) and (2S)-phenyl[2,3-2H]-alanine. 3. By means of 1H nuclear magnetic resonance spectroscopy and the action of D and L-amino acid oxidase the configuration of the phenylalamine has been shown to be (2R, 3S) and (2S, 3S), respectively. The labelled phenylalanine is thus sterically and isotopically homogenous at position 3 but heterogenous at position 2.     

The stereochemical course of thiophosphoryl group transfer catalyzed by adenosine kinase

Adenosine kinase was partially purified form beef liver and used to catalyze the conversion of (γR)ATPγS,γ¹⁸O and adenosine to ADP and AMPαS,α¹⁸O. The configuration at phosphorus in AMPαS,α¹⁸O was established by subjecting it to stereospecific phosphorylation to (αS)ATPαS,α¹⁸O and showing that only the nonbridging oxygen bonded to the α-P was enriched with ¹⁸O. The configuration at α-P in AMPαS,α¹⁸O was therefore S, and the transfer of the [¹⁸O]thiophosphoryl group occurred with $\text{inversion}$ of configuration.     

Mechanism of self-phosphorylation of adenosine 3':5'-monophosphate-dependent protein kinase from bovine cardiac muscle.

The adenosine 3':5'-monophosphate (cAMP)-dependent protein kinase purified from bovine cardiac muscle catalyzes the transfer of up to 2 mol of 32P from [lambda-32P]ATP to seryl residues in its cyclic nucleotide-binding protein component (Erlichman, J., Rosenfeld, R., and Rosen, O. M. (1974) J. Biol. Chem. 249, 5000-5003). We now present three lines of evidence to support our conclusions that the undissociated holoenzyme does not catalyze the phosphorylation of exogenous substrates but can undergo self-phosphorylation by an intramolecular reaction: (a) addition of either cAMP-binding protein or the protein kinase inhibitor (Walsh, D. A., Ashby C. D., Gonzales, C., Calkins, D., Fischer, E. H., and Krebs, D. G. (1971) J. Biol. Chem. 241, 1977-1985) does not inhibit self-phosphorylation as it does phosphorylation of exogenous substrates in the presence or absence of cAMP; (b) addition of catalytic subunit to an excess of cyclic nucleotide-binding protein results in phosphorylation equivalent to the amount of holoenzyme so generated; (c) the rate of self-phosphorylation is not affected by dilution of the holoenzyme.     

Isolation and characterization of a unique galactoside from male Drosophila melanogaster.

A ninhydrin-positive compound with presumptive hormonal activity, previously considered to be a peptide (Chen, P.S., and Bühler, R. (1970), J. Insect Physiol. 16, 615), has been isolated from adult male Drosophila melanogaster. Chromatographic analysis of the acid-hydrolyzed material revealed the presence of ethanolamine, phosphorus, galactose, and glycerol. Chemical analysis showed these to be present in equimolar amounts. Based on its phosphorus content, the nonreducing material took up 2 equiv of periodate, and released 1 equiv of formaldehyde. Characterization of the compound as 1-O-(4-O-(2-aminoethyl phosphate)-beta-D-galactopyranosyl)-x-glycerol was achieved by gas chromatography-mass spectroscopy and 1H and 31P NMR using model compounds. In vivo synthesis from labeled precursors is in accord with the proposed structure.     

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.     

A new synthesis of adenosine 5'-([gamma(R)-17O,18O]-gamma-thiotriphosphate) and its use to determine the stereochemical course of the activation of glutamate by glutamine synthetase

A new synthetic route to adenosine 5'-([gamma(R)-17O,18O]-gamma-thiotriphosphate) is described which combines chemical methods for introducing the heavy oxygen isotopes and enzymic methods for achieving the enantiospecificity. This material was used as a substrate for the activation of glutamate catalyzed by glutamine synthetase from Salmonella typhimurium. Analysis of the chirality of the [16O,17O,18O]thiophosphate produced showed that the reaction proceeds with inversion of configuration on phosphorus. This result, taken together with the positional isotope exchange studies of Midelfort and Rose [Midelfort, C. F., & Rose, I.A. (1976) J. Biol. Chem. 251, 5881-5887], demonstrates that the activation of glutamate to form gamma-glutamyl phosphate proceeds by a direct "in-line" transfer of the phosphoryl group.