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.     

The stereochemical course of phosphoric residue transfer catalyzed by sarcoplasmic reticulum ATPase

The stereochemical course of the phosphoric residue transfer from ADP to water catalyzed by the (Mg2+ + Ca2+)-dependent ATPase of sarcoplasmic reticulum has been determined. For this determination, the preparation is described of ATP gamma S, stereospecifically labeled in the gamma-position with both 17O and 18O. After hydrolysis of this nucleotide, the analysis of the product inorganic [16O,17O,18O]thiophosphate showed that the reaction proceeded with retention of configuration at the gamma-phosphorus atom. This result is expected since a phosphoenzyme is well characterized for this ATPase and provides support for the hypothesis that each phosphate transfer step occurs with inversion. In this case, the formation and breakdown of the phosphoenzyme occur each with inversion leading to the retention observed for the whole reaction.     

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 phosphoric residue transfer catalyzed by beef heart mitochondrial ATPase

The stereochemical course of phosphoric residue transfer has been determined for beef heart mitochondrial ATPase. When aden 5'-(3-thiotriphosphate), stereospecifically labeled with 18O in the gamma position, was hydrolyzed in [17O]water in the presence of the ATPase, the product inorganic [16O, 17O, 18O]thiophosphate was chiral. The configuration of the product showed that the hydrolysis had proceeded with inversion at the gamma-phosphorus atom. This result suggests that there is a direct, in-line transfer of the phosphoric residue between ADP and water and that there is no phosphoenzyme intermediate.     

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.     

The stereochemical course of phosphoryl transfer catalysed by glucokinase.

Adenosine 5'-[gamma(S)-16O,17O,18O]triphosphate has been used to determine the stereo-chemical course of phosphoryl transfer catalysed by rat liver glucokinase. The chirality of the product, D-glucose 6-[16O,17O,18O]phosphate was analysed by 31P n.m.r. spectroscopy. The reaction proceeds with inversion of configuration at phosphorus. The simplest interpretation of this result, which is the same as that observed with yeast hexokinase [Lowe & Potter (1981) Biochem. J. 199, 277-233], is that the phosphoryl group is transferred between MgATP2- and glucose in the ternary complex by an 'in-line' mechanism. It accords with the veiw that the kinetic differences between glucokinase and the other hexokinases arise from differences in rate constants and not from any fundamental differences in chemical mechanism.     

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.     

The stereochemical course of phosphoryl transfer catalysed by glucose 6-phosphatase.

Rat liver microsomal glucose 6-phosphatase catalyses phosphoryl transfer between D-glucose 6-[(R)-16O,17O,18O]phosphate and D-glucose with retention of configuration at the phosphorus atom. Since individual phosphoryl-transfer steps appear in general to occur with inversion of configuration, this observation is most simply interpreted in terms of a double-displacement mechanism with a phosphoryl-enzyme intermediate. Such an intermediate has been proposed previously from kinetic and 32P-labelling experiments.     

Mechanism and stereochemical course at phosphorus of the reaction catalyzed by a bacterial phosphotriesterase

The reaction mechanism for the phosphotriesterase from Pseudomonas diminuta has been examined. When paraoxon (diethyl 4-nitrophenyl phosphate) is hydrolyzed by this enzyme in oxygen-18-labeled water, the oxygen-18 label is found exclusively in the diethyl phosphate product. The absolute configurations for the (+) and (-) enantiomers of O-ethyl phenylphosphonothioic acid have been determined by X-ray diffraction structural determination of the individual crystalline 1-phenylethylamine salts. The (+) enantiomer of the free acid corresponds to the RP configuration. The RP enantiomer of O-ethyl phenylphosphonothioic acid has been converted to the SP enantiomer of EPN [O-ethyl O-(4-nitrophenyl) phenylphosphonothioate]. (SP)-EPN is hydrolyzed by the phosphotriesterase to the SP enantiomer of O-ethyl phenylphosphonothioic acid. The enzymatic reaction therefore proceeds with inversion of configuration. These results have been interpreted as an indication of a single in-line displacement by an activated water molecule directly at the phosphorus center of the phosphotriester substrate. (RP)-EPN is not hydrolyzed by the enzyme at an appreciable rate.     

GTPase-mediated activation of ATP sulfurylase.

GTP stimulates the synthesis of APS (adenosine 5'-phosphosulfate) by the enzyme ATP sulfurylase (ATP:sulfate adenylyltransferase, EC 2.7.7.4) via a GTPase mechanism. The activation of the enzyme, purified from Escherichia coli, is titratable with GTP. The initial rate of APS formation is increased 116-fold at a saturating concentration of GTP. The enzyme exhibits a GTPase activity that is stimulated by ATP and further enhanced by SO4; however, SO4 alone does not significantly stimulate GTP hydrolysis. The larger subunit of ATP sulfurylase, encoded by cysN, contains a GTP-binding consensus sequence common to other known GTP-binding proteins. This is the first evidence that the sulfate activation pathway is a metabolic target for regulation by a GTPase.     

The stereochemical course of phosphoric residue transfer during the myosin ATPase reaction

When adenosine 5'-(3-thiotriphosphate), stereospecifically labeled in the gamma position with 18O, was hydrolyzed in the presence of myosin subfragment 1 in 17O-enriched water, the product inorganic [16O,17O,18O]thiophosphate was chiral. The configuration of this product showed that the hydrolysis proceeds with inversion at the transferred phosphoric residue. This result suggests a direct, in-line hydrolysis mechanism for the ATPase.     

Stereochemical course of thiophosphoryl transfer catalyzed by cytosolic phosphoenolpyruvate carboxykinase

Rat liver cytosolic phosphoenolpyruvate carboxykinase (PEPCK) utilizes inosine 5'-(3-thiotriphosphate) (ITP gamma S) as an excellent substrate, with Km and V values of 0.08 mM and 37 mumol min-1 (mg of protein)-1, respectively, compared with the corresponding values of 0.168 mM and 76 mumol min-1 (mg of protein)-1 for ITP. Thus, the V/Km values for the two substrates are the same. Reaction of (RP)-[gamma-18O2]ITP gamma S with oxalacetate catalyzed by cytosolic PEPCK produces (SP)-thio[18O]phosphoenolpyruvate. Therefore, thiophosphoryl transfer catalyzed by this enzyme proceeds with overall inversion of configuration at P. The reaction mechanism involves an uneven number of phosphotransfer steps, most likely a single step transfer between bound substrates. The results do not support the involvement of a phosphoryl enzyme intermediate in the mechanism.     

Stereochemical course of thiophosphoryl group transfer catalyzed by mitochondrial phosphoenolpyruvate carboxykinase

Guinea pig liver mitochondrial phosphoenolpyruvate carboxykinase catalyzes the conversion of (Rp)-guanosine 5'-(3-thio[3-18O]triphosphate) and oxalacetate to (Sp)-[18O] thiophosphoenolpyruvate , GDP, and CO2 by a mechanism that involves overall inversion in the configuration of the chiral [18O]thiophosphate group. This result is most consistent with a single displacement mechanism in which the [18O]thiophosphoryl group is transferred from (Rp)-guanosine 5'-(3-thio[3-18O]triphosphate) bound at the active site directly to enolpyruvate generated at the active site by the decarboxylation of oxalacetate. In particular, this result does not indicate the involvement of a covalent thiophosphoryl-enzyme on the reaction pathway.     

Kinetic mechanism of the Mg2+-dependent nucleotidyl transfer catalyzed by T4 DNA and RNA ligases.

The Mg(2+)-dependent adenylylation of the T4 DNA and RNA ligases was studied in the absence of a DNA substrate using transient optical absorbance and fluorescence spectroscopy. The concentrations of Mg(2+), ATP, and pyrophosphate were systematically varied, and the results led to the conclusion that the nucleotidyl transfer proceeds according to a two-metal ion mechanism. According to this mechanism, only the di-magnesium-coordinated form Mg(2)ATP(0) reacts with the enzyme forming the covalent complex E.AMP. The reverse reaction (ATP synthesis) occurs between the mono-magnesium-coordinated pyrophosphate form MgP(2)O(7)(2-) and the enzyme.MgAMP complex. The nucleotide binding rate decreases in the sequence ATP(4-) > MgATP(2-) > Mg(2)ATP(0), indicating that the formation of the non-covalent enzyme.nucleotide complex is driven by electrostatic interactions. T4 DNA ligase shows notably higher rates of ATP binding and of subsequent adenylylation compared with RNA ligase, in part because it decreases the K(d) of Mg(2+) for the enzyme-bound Mg(2)ATP(0) more than 10-fold. To elucidate the role of Mg(2+) in the nucleotidyl transfer catalyzed by T4 DNA and RNA ligases, we propose a transition state configuration, in which the catalytic Mg(2+) ion coordinates to both reacting nucleophiles: the lysyl moiety of the enzyme that forms the phosphoramidate bond and the alpha-beta-bridging oxygen of ATP.     

The stereochemical course of acetate activation by yeast acetyl-CoA synthetase.

The purified alpha-thiophosphate diastereoisomers of adenosine 5'-(1-thio)-triphosphate were used to study the stereochemical course of the reaction catalyzed by yeast acetyl-CoA synthetase. Asymmetrically labeled adenosine 5'-thiophosphate was formed from the "B" diastereoisomer of adenosine 5'-(1-thio)-triphosphate and [18O]acetate. The label was found to be in the opposite orientation from the leaving pyrophosphate group showing that the acetate activation step occurred with inversion of configuration at the alpha-phosphorus.     

Kinetic mechanism of the dimeric ATP sulfurylase from plants

In plants, sulfur must be obtained from the environment and assimilated into usable forms for metabolism. ATP sulfurylase catalyses the thermodynamically unfavourable formation of a mixed phosphosulfate anhydride in APS (adenosine 5′-phosphosulfate) from ATP and sulfate as the first committed step of sulfur assimilation in plants. In contrast to the multi-functional, allosterically regulated ATP sulfurylases from bacteria, fungi and mammals, the plant enzyme functions as a mono-functional, non-allosteric homodimer. Owing to these differences, here we examine the kinetic mechanism of soybean ATP sulfurylase [GmATPS1 (Glycine max (soybean) ATP sulfurylase isoform 1)]. For the forward reaction (APS synthesis), initial velocity methods indicate a single-displacement mechanism. Dead-end inhibition studies with chlorate showed competitive inhibition versus sulfate and non-competitive inhibition versus APS. Initial velocity studies of the reverse reaction (ATP synthesis) demonstrate a sequential mechanism with global fitting analysis suggesting an ordered binding of substrates. ITC (isothermal titration calorimetry) showed tight binding of APS to GmATPS1. In contrast, binding of PP_i (pyrophosphate) to GmATPS1 was not detected, although titration of the E•APS complex with PP_i in the absence of magnesium displayed ternary complex formation. These results suggest a kinetic mechanism in which ATP and APS are the first substrates bound in the forward and reverse reactions, respectively.     

The stereochemical course of 4-hydroxy-2-nonenal metabolism by glutathione S-transferases

4-Hydroxy-2-nonenal (HNE) is a toxic aldehyde generated during lipid peroxidation and has been implicated in a variety of pathological states associated with oxidative stress. Glutathione S-transferase (GST) A4-4 is recognized as one of the predominant enzymes responsible for the metabolism of HNE. However, substrate and product stereoselectivity remain to be fully explored. The results from a product formation assay indicate that hGSTA4-4 exhibits a modest preference for the biotransformation of S-HNE in the presence of both enantiomers. Liquid chromatography mass spectrometry analyses using the racemic and enantioisomeric HNE substrates explicitly demonstrate that hGSTA4-4 conjugates glutathione to both HNE enantiomers in a completely stereoselective manner that is not maintained in the spontaneous reaction. Compared with other hGST isoforms, hGSTA4-4 shows the highest degree of stereoselectivity. NMR experiments in combination with simulated annealing structure determinations enabled the determination of stereochemical configurations for the GSHNE diastereomers and are consistent with an hGSTA4-4-catalyzed nucleophilic attack that produces only the S-configuration at the site of conjugation, regardless of substrate chirality. In total these results indicate that hGSTA4-4 exhibits an intriguing combination of low substrate stereoselectivity with strict product stereoselectivity. This behavior allows for the detoxification of both HNE enantiomers while generating only a select set of GSHNE diastereomers with potential stereochemical implications concerning their effects and fates in biological tissues.