18O-labelled Water Research Articles

Unveiling novel pathways and key contributors in the nitrogen cycle: Validation of enrichment and taxonomic characterization of oxygenic denitrifying microorganisms in environmental samples

Microorganisms play a crucial role in both the nitrogen cycle and greenhouse gas emissions. A recent discovery has unveiled a new denitrification pathway called oxygenic denitrification, entailing the enzymatic reduction of nitrite to nitric oxide (NO) by a putative nitric oxide dismutase (nod) enzyme. In this study, the presence of the nod gene was detected and subsequently enriched in anaerobic-activated sludge, farmland soil, and paddy soil samples. After 150 days, the enriched samples exhibited significant denitrification, and concomitant oxygen production. The removal efficiency of nitrite ranged from 64.6 % to 79.0 %, while the oxygen production rate was between 15.4 μL/min and 18.6 μL/min when exposed to a sole nitrogen source of 80 mg/L sodium nitrite. Additionally, batch experiments and kinetic analyses revealed the intricate pathways and underlying mechanisms governing the oxygenic denitrification reaction by using CARBOXY-PTIO, 18O-labelled water, and acetylene to unravel the intricacies of the reaction. The quantitative polymerase chain reaction (qPCR) results indicated a significant surge in the abundance of nod genes, escalating from 7.59 to 10.12-fold. Moreover, analysis of 16S ribosomal DNA (rDNA) amplicons revealed Proteobacteria as the dominant phylum and Thauera as the main genus, with the presumed affiliation. In this study, a new nitrogen conversion pathway, oxygenic denitrification, was discovered in environmental samples. This process provides the possibility for the control of nitrous oxide in the treatment of nitrogenous wastewater.

Deforestation for agriculture increases microbial carbon use efficiency in subarctic soils

Agriculture is likely to expand poleward with climate change, encouraging deforestation for agriculture in subarctic regions, which alters soil physical, chemical and biological properties and potentially affects microbial metabolic efficiency. Deciphering how and by which mechanisms land-use change affects microbial carbon use efficiency (CUE) will enable the development of mitigation strategies to alleviate C losses. We assessed CUE using 18O-labelled water in a paired-plot approach on soils collected from 19 farms across the subarctic region of Yukon, Canada, comprising 14 pairs of forest-to-grassland conversion and 15 pairs of forest-to-cropland conversion. Microbial CUE significantly increased following conversion to grassland and cropland. Land-use conversion resulted in a lower estimated abundance of fungi, while the archaeal abundance increased. Interestingly, structural equation modelling revealed that increases in CUE were mediated by a rise in soil pH and a decrease in soil C:N ratio rather than by shifts in microbial community composition, i.e. the ratio of fungi, bacteria and archaea. Our findings indicate a direct control of abiotic factors on microbial CUE via improved nutrient availability and facilitated conditions for microbial growth. Overall, this implies that to a certain extent CUE can be managed to achieve a more efficient build-up of stabilised soil organic C (SOC), as reflected in increased mineral-associated organic C under agricultural land use. These insights may also help constrain SOC models that generally struggle to predict the effects of deforestation, something that is likely to take place more frequently in the subarctic.

Oxygen gas derived oxygen does not affect the accuracy of 18O-labelled water approach for microbial carbon use efficiency

The 18O-labelled-water incubation approach is the latest widely used method for soil microbial carbon use efficiency (CUE) estimation. This approach assumes oxygen used for microbial DNA synthesis is from growth medium water. However, whether the oxygen might be from oxygen gas (O2) is unknown. Here we compared the results of 18O-labelled water and 18O-labelled O2 incubation experiments following the standard procedure of soil microbial CUE measurement. Results indicated that 99.25% of the oxygen atoms in the newly synthesized microbial DNA after 48 h of incubation were derived from applied water, while 0.75% of the oxygen atoms were derived from O2. Our study suggests the contribution of O2 to microbial DNA synthesis is negligible during short-term incubation and the 18O-labelled-water approach is reliable for estimating soil microbial CUE for the tested forest soil.

Evaluation of the 18O-H2O incubation method for measurement of soil microbial carbon use efficiency

The 18O-labelled water approach has been widely used to estimate microbial carbon use efficiency (CUE). However, this approach assumes that oxygen in microbial DNA synthesis is 100% from water while this assumption has not been tested. We tested this assumption using 18O-labelled glucose and 18O-labelled water to see their contributions to the oxygen in microbial DNA synthesis. Results showed microbes used less than 8.3% glucose-oxygen and more than 91.7% water-oxygen for their DNA synthesis. The 18O-labelled water method for estimation of CUE has proven to be reliable under low enrichment of 18O in soil water. We suggest to use a correction factor (i.e., 1.1 in this study) to account for oxygen incorporated into DNA from other sources than water.

Suitability of precipitation waters as semi-artificial groundwater tracers

In this study the suitability of precipitation waters as semi-artificial and multi-component groundwater tracers is investigated and potential applications as well as limitations are discussed. Specifically, we analyzed their migration behavior during laboratory-scale experiments with groundwater-saturated porous media. Artificially 2H- and 18O-labelled water as well as natural precipitation waters were injected into sediment-packed columns and traced via stable isotope analysis. Their migration behavior was compared to that of widely used tracers. These flow-through experiments were underpinned by a batch reactor study and by hydrogeochemical modeling. While most artificial isotope applications showed a high stability and conservative transport behavior for all tested sediments, the breakthrough curves observed in some experiments with rain and snowmelt were unsteady. This was most likely caused by the small ratio between the tracer’s isotopic difference compared to the background signal and the available analytical precision. The minimum ratio required for effectively reducing uncertainties during data evaluation showed to depend significantly on experimental conditions but should not decrease below ∼10 during peak breakthrough and preferably be higher. Furthermore, our study illustrates that measurements based on using the precipitation waters’ inherent low electrical conductivity can be significantly biased by water-sediment reactions. The batch study and reactive transport simulations confirmed this observation while revealing mineral reactions and, to a minor extent, also ion exchange as the underlying processes.

Biopore effects on phosphorus biogeochemistry in subsoils

Biopores are characterised by high concentrations of plant available nutrients and provide preferential pathways for root growth into the subsoil, thereby potentially enabling plants to access phosphorus (P) resources located in the subsoil. Here, we sampled biopores from a replicated agricultural field trial in Klein-Altendorf, Germany, to analyse their nutrient composition and P speciation as determined by Hedley sequential extraction and X-ray absorption near edge structure (XANES) spectroscopy. In addition, we analysed the oxygen isotopic composition of HCl P (δ18OHClP) as an indicator of long-term effects of biological P turnover. We found that biopore effects were most pronounced in the subsoil, where the concentration of easily extractable (labile) P tended to be greater in biopores than in bulk soil, as evident in both Hedley sequential extraction and XANES spectroscopy. We assume that these findings result from inputs of organic matter from the topsoil as well as an input of Ca-particles into subsoil biopores by earthworm activity. Biologically cycled P was subsequently precipitated as Ca-P as evident by δ18OHClP values close to equilibrium in biopores even at great depths. When incubating bulk soil samples with 18O-labelled water, however, we observed a significant increase of δ18OHClP values in the topsoil, but only small if any changes of δ18OHClP values in the subsoil. Thus, biopores present hotspots of P cycling in the subsoil, but the effect of biopores on overall P turnover in the bulk subsoil is limited.

A new method to identify hydrolytic degradants in drug substances with UPLC-MS using 18O-labelled water

Drug substances are susceptible to many different degradation pathways due to diverse nature of the molecule structures. Hydrolysis is one of the most common reactions seen for drug substances and drug products since water is ubiquitously present either as moisture or included in some formulations such as parenteral products. The hydrolytic degradation of fluticasone propionate, duloxetine and amlodipine was studied in this work using 18O-labelled water with UPLC-MS. The structural proposals of the degradants are based solely on the molecular mass of the degradants with enhanced certainty by corresponding results from 18O labeling experiments. The advantage of using 18O labeling experiments is the extra information from knowing whether a water molecule is incorporated into a degradant or not, therefore enabling either confirmation or rejection of a proposed molecular structure.

Electrochemical Reaction of Diphenylacetaldehyde in the Presence of Alcohols: Successive Reactions at the Anode and Cathode

We recently described the anodic oxidation of 1,1-diphenylacetaldehyde (1) to benzophenone in the presence of water.1 The reaction was postulated to involve nucleophilic attack by water upon a cationic intermediate to afford alpha-hydroxydiphenylacetaldehyde, which then undergoes anodic decarbonylation to afford benzophenone. This hypothesis was tested by carrying out the electrolysis in 18O-labelled water. As expected, the benzophenone product was found to have incorporated the 18O label. We then anticipated that substitution of an alcohol (2) for water in the electrolysis of 1 would produce an a-alkoxyaldehyde (3) that would be more resistant to subsequent oxidation. We describe here the results of a study confirming this expectation but revealing further unsuspected mechanistic complexity, Electrochemical reaction in acetonitrile of diphenylacetaldehyde (1) in a undivided cell containing two carbon electrodes in the presence of cyclohexanol under constant current conditions affords alpha-cyclohexyloxydiphenylacetaldehyde cyclohexanol hemiacetal (2, R = cyclohexyl), which then ejects cyclohexanol to afford the corresponding aldehyde (3). Upon continued electrolysis, 3 is converted to a benzhydryl alkyl ether 4(Scheme 1). Scheme 1. (insert image here) The most surprising and indeed unprecedented feature of the electrochemistry of 1 under these conditions is the fact that both electrodes are involved. Since 1 is converted to 3 at the anode via 2, we initially assumed that the ethers 4 are produced by a succeeding anodic oxidation of 3.However, two control experiments disproved this hypothesis. First, 3 (independently synthesized by reaction of alpha-bromo-diphenylacetaldehyde2 with cyclohexanol in the presence of silver tetrafluoroborate3) was placed in the anode compartment of a divided cell. It was found to be stable to anodic oxidation. On the other hand, when it was placed in the cathode compartment, it was quickly decarbonylated to 4a .The mechanism of this unusual mild cathodic cleavage reaction is under investigation.Baizer introduced the term ‘paired electrolyses” for processes in which both electrodes participate.4 Examples include (a) parallel electrolyses, in which two components of a solution undergo simultaneous independent and unrelated transformation;5 (b) generation of two reagents, one at each electrode, that can react with a third component of the medium, as in the conversion of ethylene to propylene oxide by the dual action of electrogenerated hydroxide and bromine;6 or the simultaneous oxidation of glucose to gluconic acid at the anode and reduction to sorbitol at the cathode.7 Conversion of 1a to ethers 4 is, however, the first example of an organic electrode reaction involving significant structural transformations of a substance at both electrodes, that is, not merely changes in oxidation state, such as in the anodic oxidation of t-butylphenol to t-butylquinone followed by cathodic reduction of the latter to the hydroquinone.8 REFERENCES(1) Merzel, R. L.; Fry, A. J. J. Electrochem. Soc. 2012, 159. (2) Padwa, A.; Dehm, D. J. Org. Chem. 1975, 40, 3139.(3) Fry, A. J.; Migron, Y. Tetrahedron Lett. 1979, 20, 3357.(4) Baizer, M. M. in Lund, H. Baizer, M. M., eds., Organic Electrochemistry, 3rd ed., Dekker: New York, N.Y., p. 1421. (5) Baizer, M. M.; Hallcher, R. C. J. Electrochem. Soc. 1976, 123, 809. (6) Manji, A.; Oloman, C. W. J. Appl. Electrochem. 1987, 17, 532.(7) Pintauro, P. N.; Johnson, D. K.; Park, K.; Baizer, M. M.; Nobe, K. J. Appl. Electrochem. 1984, 14, 209.(8) Ref. 4, Table 1 and references therein.

A new mechanistic model of δ18O-N2O formation by denitrification

Anaerobic incubations of flooded and non-flooded agricultural soils and stream sediment were conducted with 18O-labelled water to investigate the stable isotope ratios (δ15N and δ18O) of nitrous oxide (N2O) produced from denitrification. The rates of N2O production and δ15N- and δ18O-N2O values were measured. The amount of oxygen exchange (O-exchange) with water, and the nitrogen and oxygen isotope effects (ε) were calculated. The net 15N isotope effect (NO3−→N2O) for denitrification in this study varied from −30‰ to −9‰. The net 18O isotope effect ranged between +32‰ and +60‰ and was negatively correlated to the total fraction of O-exchange, which varied between 0.40 and 0.94.This manuscript describes a new, comprehensive set of mathematical expressions that can be used to model δ18O values of N2O formed by denitrification and calculate the magnitude of O-exchange and the net 18O isotope effect for denitrification. This mathematical approach is compared to another method of approximating O-exchange (Snider et al., 2009), and we show that this older method provides a minimum estimate of O-exchange. Using this mechanistic model, we discuss how N2O consumption, open/closed systems, and variations in the N2O:N2 ratio can influence the observed δ18O-N2O.The net 18O isotope effect for denitrification in this study was partially controlled by the fractions of O-exchange and N2O reduction, which were likely influenced by the actively denitrifying microbial community and the soil moisture. We conclude that δ18O-N2O values are, in many cases, useful tracers of N2O production because they are often higher than the majority of nitrification-derived δ18O-N2O values. The usefulness of δ18O values to apportion sources must be determined on a case-by-case basis.

Novel insights in the study of water penetration into polycrystalline UO 2 by secondary ion mass spectrometry

Oxygen and water species (e.g., OH −, H 2O) diffusion in polycrystalline UO 2 are key parameters to investigate in order to predict spent fuel behaviour in a final repository in the event of exposure to groundwater. In this work, 18O tracer diffusion is evaluated by means of secondary ion mass spectrometry on a UO 2 pellet exposed to 18O-labelled water at room temperature for 3 months. Depth profiling up to 22 μm beneath the pellet surface clearly indicates a combination of oxygen diffusion into the UO 2 lattice and water species diffusion along grain boundaries, behaving as high-diffusivity-paths. The relevant coefficients have been estimated around 1.4 × 10 −24 m 2 s −1 for lattice diffusion and around 2.3 × 10 −16 m 2 s −1 for grain boundary diffusion.

Characterization of the Penetration Mechanisms of Water into Polycrystalline UO2

AbstractFollowing containment failure in the scenario of geological disposal of spent nuclear fuel, the penetration rate of groundwater into the UO2 matrix could cause a rapid increase of the fraction of inventory becoming available for prompt dissolution. In this respect, oxygen and water diffusion mechanisms are key issues to investigate. In this work, secondary-ion-mass-spectrometry (SIMS) depth profiling has been applied to characterize a polycrystalline UO2 pellet exposed to 18O-labelled water at room temperature. 18O depth profiling up to 25 μm beneath the pellet surface clearly indicates a combination of oxygen diffusion into the UO2 lattice and water diffusion along grain boundaries, behaving as high diffusivity paths. The lattice diffusion coefficient of oxygen, DL, and the quantity δDB – product of the grain boundary width, δ, and the grain boundary diffusion coefficient of water, DB – have been measured, resulting in DL = (2.5 ± 0.1) × 10-24 m2 s-1 and δDB = (7.5 ± 0.3) × 10-24 m3 s-1.

Pleurotus species convert monoterpenes to furanoterpenoids through 1,4-endoperoxides

Enzymatic synthesis of furanoterpenoids from β-myrcene and related monoterpenes was observed using a solubilised enzyme fraction of mycelium lyophilisates of several Pleurotus species. The initial enzymatic step, the incorporation of molecular oxygen into the conjugated 1,3-diene moiety, was similar to a 2 + 4 cycloaddition of 1,3-dienes with dienophilic 1O 2, and was followed by a non-catalysed degradation sequence leading to the furans. The cyclic peroxides 3,6-dihydro-4-(2-(3,3-dimethyloxiran-2-yl)ethyl)-1,2-dioxine and 5-(3,6-dihydro-1,2-dioxin-4-yl)-2-methylpentan-2-ol were identified as key intermediates. Biotransformation of β-myrcene in 18O-labelled HEPES-buffer did not yield a detectable label in perillene, so a water addition to 3,10-epoxy-β-myrcenes as an alternative was ruled out. The pathway suggested presents a substantiated biogenetic scheme for the formation of monoterpenoid furans and opens biotechnological access to valuable natural flavour compounds, such as perillene and rosefurane. Only one metabolite, identified as the new natural compound 6-methyl-2-methylene-hept-5-enal, carried the 18O-label. The enzymatic formation of this compound through a 1,2-endoperoxide (3-(5-methyl-1-methylene-hex-4-enyl)-[1,2]-dioxetane) is suggested. The label may simply result from a chemical oxygen exchange between the carbonyl group and the 18O-labelled water.

Chemical Kinetics and Aqueous Degradation Pathways of a New Class of Synthetic Ozonide Antimalarials

Chemical stability of a new class of ozonide (1,2,4 trioxolanes) antimalarial compounds was investigated. The effects of pH, ionic strength, dielectric constant and cyclodextrin-complexation on the chemical stability and degradation product formation of selected compounds were examined. The mechanism of degradation in aqueous solution was probed using 18O-labelled water and kinetic solvent isotope effect studies. The effect of stereochemistry was investigated using selected pairs of stereoisomers. The degradation of the ozonides in aqueous solution followed apparent first-order kinetics, with no effect of ionic strength and no indication of any direct involvement of water in the degradation mechanism. All major degradation products were identified and mass balance was confirmed. Stereochemistry had a significant effect on degradation rate; trans isomers degrading approximately four-fold faster than the corresponding cis isomers. The degradation rates were essentially independent of pH above pH 2; however, an additional specific acid catalysed pathway was dominant below pH 2. Solvent dielectric constant had a significant effect on the degradation rate. It is proposed that the degradation observed in aqueous solution occurred through a concerted heterolytic scission of the central ozonide ring, with chemical substituents on the cyclohexyl ring having only a minor influence on degradation rate.

A novel dual‐isotope labelling method for distinguishing between soil sources of N2O

We present a novel 18O-15N-enrichment method for the distinction between nitrous oxide (N2O) from nitrification, nitrifier denitrification and denitrification based on a method with single- and double-15N-labelled ammonium nitrate. We added a new treatment with 18O-labelled water to quantify N2O from nitrifier denitrification. The theory behind this is that ammonia oxidisers use oxygen (O2) from soil air for the oxidation of ammonia (NH3), but use H2O for the oxidation of the resulting hydroxylamine (NH2OH) to nitrite (NO2-). Thus, N2O from nitrification would therefore be expected to reflect the 18O signature of soil O2, whereas the 18O signature of N2O from nitrifier denitrification would reflect that of both soil O2 and H2O. It was assumed that (a) there would be no preferential removal of 18O or 16O during nitrifier denitrification or denitrification, (b) the 18O signature of the applied 18O-labelled water would remain constant over the experimental period, and (c) any O exchange between H(2)18O and NO3- would be negligible under the chosen experimental conditions. These assumptions were tested and validated for a silt loam soil at 50% water-filled pore space (WFPS) following application of 400 mg N kg-1 dry soil. We compared the results of our new method with those of a conventional inhibition method using 0.02% v/v acetylene (C2H2) and 80% v/v O2 in helium. Both the 18O-15N-enrichment and inhibitor methods identified nitrifier denitrification to be a major source of N2O, accounting for 44 and 40%, respectively, of N2O production over 24 h. However, compared to our 18O-15N-method, the inhibitor method overestimated the contribution from nitrification at the expense of denitrification, probably due to incomplete inhibition of nitrifier denitrification and denitrification by large concentrations of O2 and a negative effect of C2H2 on denitrification. We consider our new 18O-15N-enrichment method to be more reliable than the use of inhibitors; it enables the distinction between more soil sources of N2O than was previously possible and has provided the first direct evidence of the significance of nitrifier denitrification as a source of N2O in fertilised arable soil.

Ratios of Community Respiration to Photosynthesis and Rates of Primary Production in Lake Erie Via Oxygen Isotope Techniques

To evaluate levels of primary production and community metabolism in Lake Erie, we conducted incubations with 18O-labelled water and determined the ratio of respiration to primary production (R:P) during the summer and early fall of 2002. The epilimnion of Lake Erie was characterized by δ 18O values less than 0.7‰ at all times that reflects a strong contribution of O 2 from primary production. High δ 18O values (maximum of 6.6‰) were common in the O 2 depleted waters of the hypolimnion as a consequence of isotope fractionation during respiration. Hypoxic conditions were evident in the central basin in August when the fractional abundance of O 2 reached a minimum value of 0.04. Rates of primary production varied from 0.23 to 1.76 mmol-O 2.m –3.h –1, decreased from west to east in August, and were at a minimum in the central basin in September. Ratios of respiration to photosynthesis (R:P) (determined from the abundance and isotopic composition of dissolved O 2) in the epilimnion as low as 0.45 in July were strong evidence of net autotrophy and preceded the development of hypoxia in August. Net heterotrophy prevailed in August and September. The absolute values of R:P ratios were not indicative of trophic state, however, the wide range of R:P ratios (0.45 to 20.40), fraction of O 2 saturation (0.04 to 1.36), and δ 18O-O 2 (−6.0 to 7.5‰) values were all indicative of a eutrophic system. An average isotope fractionation factor for respiration of 7.9‰ was determined from samples in the hypolimnion in July and August. Based on unique fractionation factors for respiration in the water column (23.5‰) and in sediments (3‰) we calculate that 61% of hypolimnion O 2 respiration occurs within sediments and 39% occurs within the water column.

重酸素水を用いた非イオン系界面活性剤の微生物分解試験法の開発

In order to investigate the oxidative biodegradation mechanisms of octylphenol polyethoxylate (OPEO) by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS), fundamental conditions for the bacterial biodegradation test method using 18O-labelled water as an incubation medium were examined. Since 18O-labelled water is a valuable reagent, reduction of incubation medium down to 200 µL could be achieved. Furthermore, to recover OPEO degradation intermediates from a minimum amount of the medium containing basal salt, a simple solid-liquid extraction procedure was introduced. The combination of the biodegradation test using 18O-labelled water and MALDI-MS measurement thus could provide a conclusive biodegradation mechanism by the observation of incorporation of 18O atoms into the carboxyl group of OPEC during the biodegradation process.

Protein phosphatase 1 catalyses the direct hydrolytic cleavage of phosphate monoester in a ternary complex mechanism

The catalytic subunit of the Ser/Thr protein phosphatase 1 (PP1 cat) hydrolyses N-acetyl Arg-Arg-Ala-phosphoThr-Val-Ala ( K M=3.7 mM) in a reaction that is inhibited competitively by inorganic phosphate (P i, K i=1.6 mM) but unaffected by the product peptide alcohol at concentrations up to 3 mM. The enzyme does not catalyse the incorporation of 18O-label from 18O-labelled water into P i whether, or not, the product alcohol is present. The dephosphorylated product alcohol of phosphorylated histone, an alternative substrate for the enzyme, serves as a competitive inhibitor for phosphopeptide hydrolysis ( K i=60 μM) and co-mediates 18O-label exchange into P i in a concentration-dependent manner ( K M=64 μM). These results indicate that hydrolysis occurs through the direct attack of an activated water molecule on the phosphate ester moiety of the substrate in a ternary complex mechanism.

Origin of the oxygen atom in metalloporphyrin-catalyzed epoxidations with LiOCl as oxidant

Abstract Abstract – The origin of the oxygen atom incorporated into epoxides during the oxidation of olefins with the catalytic system Mn(TPP)Cl/LiOCl in the presence of 18O-labelled water has been investigated. One molar equivalent of H218O with respect to Li16OCl led to a 53 % 18O-incorporation within the epoxide formed, and near complete incorporation of 18O from water was achieved when no limiting amount of water was used. These results clearly indicate that the epoxidic oxygen atom arose from the high-valent metal-oxo species generated by LiOCl, but the exchange of the oxygen atom of hypochlorite with bulk water is fast, compared to the oxidation of the manganese(III) porphyrin by LiOCl. Therefore hypochlorite is not a suitable oxidant for mechanistic studies of metal-catalyzed oxygenation reactions involving high-valent metal-oxo species. Potassium monopersulfate is the adequate oxidant for such studies, because of its slow oxygen atom exchange with bulk water.

‘Oxo-hydroxo tautomerism’ as useful mechanistic tool in oxygenation reactions catalysed by water-soluble metalloporphyrins

High valent metal–oxo species have been evoked as active intermediates in many different oxidation reactions using manganese or iron porphyrin complexes as catalysts and oxygen atom donors (H2O2, PhIO, NaOCl, KHSO5, ... etc.) or dioxygen associated to a reductant as oxygen atom source. When these metalloporphyrin-catalysed oxidations are performed in water, such metal–oxo species are able to transfer an oxygen atom coming from either the oxygen source or from bulk water. This fact has been explained by the so-called oxo–hydroxo tautomerism, a mechanism involving a rapid shift of two electrons and one proton from a hydroxo ligand (electron-rich ligand formed by deprotonation of an aqua ligand) to the trans oxo species (electron-poor ligand) leading to the transformation of the hydroxo ligand into an electrophilic oxo entity on the opposite side of the initial oxo. This ‘oxo–hydroxo tautomerism’, evidenced by using 18O-labelled water, has been used as mechanistic tool to unambiguously characterize oxygen atom transfer mechanisms mediated by metal–oxo species in opposition to mechanisms related to free radical oxidation reactions.

Common-type acylphosphatase: steady-state kinetics and leaving-group dependence.

A number of acyl phosphates differing in the structure of the acyl moiety (as well as in the leaving-group pKa of the acids produced in hydrolysis) have been synthesized. The Km and Vmax values for the bovine common-type acylphosphatase isoenzyme have been measured at 25 degrees C and pH 5.3. The values of kcat differ widely in relation to the different structures of the tested acyl phosphates: linear relationships between log kcat and the leaving group pKa, as well as between log kcat/Km and the leaving-group pKa, were observed. On the other hand, the Km values of the different substrates are very close to each other, suggesting that the phosphate moiety of the substrate is the main chemical group interacting with the enzyme active site in the formation of the enzyme-substrate Michaelis complex. The enzyme does not catalyse transphosphorylation between substrate and concentrated nucleophilic acceptors (glycerol and methanol); nor does it catalyse H218O-inorganic phosphate oxygen exchange. It seems that no phosphoenzyme intermediate is formed in the catalytic pathway. Furthermore, during the enzymic hydrolysis of benzoyl phosphate in the presence of 18O-labelled water, only inorganic phosphate (and not benzoate) incorporates 18O, suggesting that no acyl enzyme is formed transiently. all these findings, as well as the strong dependence of kcat upon the leaving group pK1, suggest that neither a nucleophilic enzyme group nor general acid catalysis are involved in the catalytic pathway. The enzyme is competitively inhibited by Pi, but it is not inhibited by the carboxylate ions produced during substrate hydrolysis, suggesting that the last step of the catalytic process is the release of Pi. The activation energy values for the catalysed and spontaneous hydrolysis of benzoyl phosphate have been determined.