15N Isotope Effects Research Articles

Enzyme-catalyzed isotope equilibrium: A hypothesis to explain apparent N cycling phenomena in low oxygen environments

Increasing reports of anoxic pathways of nitrite oxidation in oxygen deficient systems challenge longstanding ideas about biogeochemical nitrogen turnover. Here, we present stable isotope data from experiments examining the nitrite-oxidizing bacterium, Nitrococcus mobilis, grown under nitrate-reducing conditions. Results confirm that N. mobilis reduces nitrate to nitrite under low oxygen, ostensibly via nitrite oxidoreductase (NXR) acting in reverse. Estimates for 15N isotope effects for NXR-based nitrate reduction ranged from 27 to 55‰, far larger than previously reported values for nitrate reduction catalyzed by nitrate reductase, while oxygen isotopes exhibited very little change. We suggest these observations are best explained by enzyme-catalyzed isotope equilibrium between nitrite and nitrate, similar to that reported for anammox and microbial carbon and sulfur cycling. Enzyme-catalyzed isotope equilibrium may play an underappreciated role in the application of stable isotopes to N cycling studies, including cycling rate measurements and biogeochemical models of N turnover. Our results underscore several considerations about N cycling in redox transition zones and emphasize the need to better understand the potential for enzyme-catalyzed isotope equilibrium in studies of the nitrogen cycle.

Intramolecular non-covalent isotope effects at natural abundance associated with the migration of paracetamol in solid matrices during liquid chromatography

Position-specific isotope analysis by Nuclear Magnetic Resonance spectrometry was employed to study the 13C intramolecular isotopic fractionation associated with the migration of organic substrates through different stationary phases chromatography columns. Liquid chromatography is often used to isolate compounds prior to their isotope analysis and this purification step potentially alters the isotopic composition of target compounds introducing a bias in the later measured data. Moreover, results from liquid chromatography can yield the sorption parameters needed in reactive transport models that predict the transport and fate of organic contaminants to in the environment. The aim of this study was to use intramolecular isotope analysis to study both 13C and 15N isotope effects associated with the elution of paracetamol (acetaminophen) through different stationary phases and to compare them to effects observed previously for vanillin. Results showed very different intramolecular isotope fractionation profiles depending on the chemical structure of the stationary phase. The data also demonstrate that both the amplitude and the distribution of measured isotope effects depend on the nature of the non-covalent interactions involved in the migration process. Results provided by theoretical calculation performed during this study also confirmed the direct link between observed intramolecular isotope fractionation and the nature of involved intermolecular interactions. It is concluded that the nature of the stationary phase through which the substrate passes has a major impact on the intramolecular isotopic composition of organic compounds isolated by chromatography methods..

Molecularly Imprinted Polymers for Compound-Specific Isotope Analysis of Polar Organic Micropollutants in Aquatic Environments.

Compound-specific isotope analysis (CSIA) of polar organic micropollutants in environmental waters requires a processing of large sample volumes to obtain the required analyte masses for analysis by gas chromatography/isotope-ratio mass spectrometry (GC/IRMS). However, the accumulation of organic matter of unknown isotopic composition in standard enrichment procedures currently compromises the accurate determination of isotope ratios. We explored the use of molecularly imprinted polymers (MIPs) for selective analyte enrichment for 13C/12C and 15N/14N ratio measurements by GC/IRMS using 1 H-benzotriazole, a typical corrosion inhibitor in dishwashing detergents, as example of a widely detected polar organic micropollutant. We developed procedures for the treatment of >10 L of water samples, in which custom-made MIPs enabled the selective cleanup of enriched analytes in organic solvents obtained through conventional solid-phase extractions. Hydrogen bonding interactions between the triazole moiety of 1 H-benzotriazole, and the MIP were responsible for selective interactions through an assessment of interaction enthalpies and 15N isotope effects. The procedure was applied successfully without causing isotope fractionation to river water samples, as well as in- and effluents of wastewater treatment plants containing μg/L concentrations of 1 H-benzotriazole and dissolved organic carbon (DOC) loads of up to 28 mg C/L. MIP-based treatments offer new perspectives for CSIA of organic micropollutants through the reduction of the DOC-to-micropollutant ratios.

Kinetic 15N-isotope effects on algal growth

Stable isotope labeling is a standard technique for tracing material transfer in molecular, ecological and biogeochemical studies. The main assumption in this approach is that the enrichment with a heavy isotope has no effect on the organism metabolism and growth, which is not consistent with current theoretical and empirical knowledge on kinetic isotope effects. Here, we demonstrate profound changes in growth dynamics of the green alga Raphidocelis subcapitata grown in 15N-enriched media. With increasing 15N concentration (0.37 to 50 at%), the lag phase increased, whereas maximal growth rate and total yield decreased; moreover, there was a negative relationship between the growth and the lag phase across the treatments. The latter suggests that a trade-off between growth rate and the ability to adapt to the high 15N environment may exist. Remarkably, the lag-phase response at 3.5 at% 15N was the shortest and deviated from the overall trend, thus providing partial support to the recently proposed Isotopic Resonance hypothesis, which predicts that certain isotopic composition is particularly favorable for living organisms. These findings confirm the occurrence of KIE in isotopically enriched algae and underline the importance of considering these effects when using stable isotope labeling in field and experimental studies.

The 15N induced isotope shift as an effective tool for the structure elucidation of 2,4 and 2,5 di-substituted thiazoles

15N isotope effects have been used for structural elucidation of the di-substituted thiazole 1-DFL 23805, a novel and potent dual CXCR1/CXCR2 inhibitor, developed by Dompé within the MedChem program. The compound and its 15N isotopologue have been synthesized according to the same proprietary procedure. The 1H and 13C NMR spectra have been measured. The isotope effects on chemical shifts and on coupling constants have been studied in detail. The collected data highlight a new perspective on the elucidation of disubstituted thiazole system, as only very few reference data have been published so far. All the results are consistent with the 2,4 regioisomery assignment.

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.

Structural and kinetic isotope effect studies of nicotinamidase (Pnc1) from Saccharomyces cerevisiae.

Nicotinamidases catalyze the hydrolysis of nicotinamide to nicotinic acid and ammonia. Nicotinamidases are absent in mammals but function in NAD(+) salvage in many bacteria, yeast, plants, protozoa, and metazoans. We have performed structural and kinetic investigations of the nicotinamidase from Saccharomyces cerevisiae (Pnc1). Steady-state product inhibitor analysis revealed an irreversible reaction in which ammonia is the first product released, followed by nicotinic acid. A series of nicotinamide analogues acting as inhibitors or substrates were examined, revealing that the nicotinamide carbonyl oxygen and ring nitrogen are critical for binding and reactivity. X-ray structural analysis revealed a covalent adduct between nicotinaldehyde and Cys167 of Pnc1 and coordination of the nicotinamide ring nitrogen to the active-site zinc ion. Using this structure as a guide, the function of several residues was probed via mutagenesis and primary (15)N and (13)C kinetic isotope effects (KIEs) on V/K for amide bond hydrolysis. The KIE values of almost all variants were increased, indicating that C-N bond cleavage is at least partially rate limiting; however, a decreased KIE for D51N was indicative of a stronger commitment to catalysis. In addition, KIE values using slower alternate substrates indicated that C-N bond cleavage is at least partially rate limiting with nicotinamide to highly rate limiting with thionicotinamide. A detailed mechanism involving nucleophilic attack of Cys167, followed by elimination of ammonia and then hydrolysis to liberate nicotinic acid, is discussed. These results will aid in the design of mechanism-based inhibitors to target pathogens that rely on nicotinamidase activity.

Deuterium isotope effects on 13C and 15N chemical shifts of intramolecularly hydrogen-bonded enaminocarbonyl derivatives of Meldrum’s and Tetronic acid

Secondary deuterium isotope effects on 13C and 15N nuclear shieldings in a series of cyclic enamino-diesters and enamino-esters and acyclic enaminones and enamino-esters have been examined and analysed using NMR and DFT (B3LYP/6-31G(d,p)) methods. One-dimensional and two-dimensional NMR spectra of enaminocarbonyl and their deuterated analogues were recorded in CDCl 3 and CD 2Cl 2 at variable temperatures and assigned. 1 J NH coupling constants for the derivatives of Meldrum’s and tetronic acids reveal that they exist at the NH-form. It was demonstrated that deuterium isotope effects, for the hydrogen bonded compounds, due to the deuterium substitution at the nitrogen nucleus lead to large one-bond isotope effects at nitrogen, 1Δ 15N(D), and two-bond isotope effects on carbon nuclei, 2ΔC(ND), respectively. A linear correlations exist between 2ΔC(ND) and 1Δ 15N(D) whereas the correlation with δNH is divided into two. A good agreement between the experimentally observed 2ΔC(ND) and calculated dσ 13C/dR NH was obtained. A very good correlation between calculated NH bond lengths and observed NH chemical shifts is found. The observed isotope effects are shown to depend strongly on Resonance Assisted Hydrogen bonding.

15N/ 14N and 18O/ 16O stable isotope ratios of nitrous oxide produced during denitrification in temperate forest soils

Anaerobic incubations of upland and wetland temperate forest soils from the same watershed were conducted under different moisture and temperature conditions. Rates of nitrous oxide (N 2O) production by denitrification of nitrate ( NO 3 - ) and the stable isotopic composition of the N 2O (δ 15N, δ 18O) were measured. In all soils, N 2O production increased with elevated temperature and soil moisture. At each temperature and moisture level, the rate of N 2O production in the wetland soil was greater than in the upland soil. The 15N isotope effect (ε) (product − substrate) ranged from −20‰ to −29‰. These results are consistent with other published estimates of 15N fractionation from both single species culture experiments and soil incubation studies from different ecosystems. A series of incubations were conducted with 18O-enriched water (H 2O) to determine if significant oxygen exchange (O-exchange) occurred between H 2O and N 2O precursors during denitrification. The exchange of H 2O–O with nitrite ( NO 2 - ) and/or nitric oxide (NO) oxygen has been documented in single organism culture studies but has not been demonstrated in soils prior to this study. The fraction of N 2O–O derived from H 2O–O was confined to a strikingly narrow range that differed between soil types. H 2O–O incorporation into N 2O produced from upland and wetland soils was 86% to 94% and 64% to 70%, respectively. Neither the temperature, soil moisture, nor the rate of N 2O production influenced the magnitude of O-exchange. With the exception of one treatment, the net 18O isotope effect (ε net) (product–substrate) ranged from +37‰ to +43‰. Most previous studies that have reported 18O isotope effects for denitrification of NO 3 - to N 2O have failed to account for the effect of oxygen exchange with H 2O. When high amounts of O-exchange occur after fractionation during reductive O-loss, the 18O-enrichment is effectively lost or diminished and δ 18O–N 2O values will be largely dictated by δ 18O–H 2O values and subsequent fractionation. The process and extent of O-exchange, combined with the magnitude of oxygen isotope fractionation at each reduction step, appear to be the dominant controls on the observed oxygen isotope effect. In these experiments, significant oxygen isotope fractionation was observed to occur after the majority of water O-exchange. Due to the importance of O-exchange, the net oxygen isotope effect for N 2O production in soils can only be determined using δ 18O–H 2O addition experiments with δ 18O–H 2O close to natural abundance. The results of this study support the continued use of δ 15N–N 2O analysis to fingerprint N 2O produced from the denitrification of NO 3 - . The utilization of 18O/ 16O ratios of N 2O to study N 2O production pathways in soil environments is complicated by oxygen exchange with water, which is not usually quantified in field studies. The oxygen isotope fractionation observed in this study was confined to a narrow range, and there was a clear difference in water O-exchange between soil types regardless of temperature, soil moisture, and N 2O production rate. This suggests that 18O/ 16O ratios of N 2O may be useful in characterizing the actively denitrifying microbial community.

Variability of Nitrogen Isotope Fractionation during the Reduction of Nitroaromatic Compounds with Dissolved Reductants

Compound-specific nitrogen isotope analysis was shown to be a promising tool for the quantitative assessment of abiotic reduction of nitroaromatic contaminants (NACs) under anoxic conditions. To assess the magnitude and variability of 15N fractionation for reactions with dissolved reductants, we investigated the reduction of a series of NACs with a model quinone (anthrahydroquinone-2,6-disulfonate monophenolate; AHQDS-) and a Fe(II)-catechol complex (1:2 Fe(II)-tiron complex; Fe(II)L2(6-)) over the pH range from 3 to 12 and variable reductant concentrations. Apparent kinetic isotope effects, AKIEN, for the reduction of four mononitroaromatic compounds by AHQDS- ranged from 1.039 +/- 0.003 to 1.045 +/- 0.002 (average +/- 1sigma), consistent with previous results for various mineral-bound reductants. 15N fractionation for reduction of 1,2-dinitrobenzene and 2,4,6-trinitrotoluene by AHQDS- and that of 4-chloronitrobenzene by Fe(II)L2(6-), however, showed substantial variability in AKIEN-values which decreased from 1.043 to 1.010 with increasing pH. We hypothesize that the isotope-sensitive and rate-limiting step of the overall NAC reduction can shift from the dehydration of substituted N,N-dihydroxyanilines (large 15N fractionation upon N-O bond cleavage) to protonation or reduction of nitroaromatic radical anions (small 15N isotope effect upon electron transfer) consistent with calculations of semiclassical 15N isotope effects. Our results imply that a quantitative assessment of NAC reduction using compound-specific isotope analysis (CSIA) might need to account for homogeneous and heterogeneous reactions separately.

Mechanistic Studies of the Flavoenzyme Tryptophan 2-Monooxygenase: Deuterium and 15N Kinetic Isotope Effects on Alanine Oxidation by an l-Amino Acid Oxidase

Tryptophan 2-monooxygenase (TMO) from Pseudomonas savastanoi catalyzes the oxidative decarboxylation of l-tryptophan during the biosynthesis of indoleacetic acid. Structurally and mechanistically, the enzyme is a member of the family of l-amino acid oxidases. Deuterium and 15N kinetic isotope effects were used to probe the chemical mechanism of l-alanine oxidation by TMO. The primary deuterium kinetic isotope effect was pH independent over the pH range 6.5-10, with an average value of 6.0 +/- 0.5, consistent with this being the intrinsic value. The deuterium isotope effect on the rate constant for flavin reduction by alanine was 6.3 +/- 0.9; no intermediate flavin species were observed during flavin reduction. The kcat/Kala value was 1.0145 +/- 0.0007 at pH 8. NMR analyses gave an equilibrium 15N isotope effect for deprotonation of the alanine amino group of 1.0233 +/- 0.0004, allowing calculation of the 15N isotope effect on the CH bond cleavage step of 0.9917 +/- 0.0006. The results are consistent with TMO oxidation of alanine occurring through a hydride transfer mechanism.

13C and 15N Isotope Effects for Conversion of l-Dihydroorotate to N-Carbamyl-l-aspartate Using Dihydroorotase from Hamster and Bacillus caldolyticus

In the pyrimidine biosynthetic pathway, N-carbamyl-L-aspartate (CA-asp) is converted to L-dihydroorotate (DHO) by dihydroorotase (DHOase). The mechanism of this important reaction was probed using primary and secondary 15N and 13C isotope effects on the ring opening of DHO using isotope ratio mass spectrometry (IRMS). The reaction was performed at three different temperatures (25, 37, and 45 degrees C for hamster DHOase; 37, 50, and 60 degrees C for Bacillus caldolyticus), and the product CA-asp was purified for analysis. The primary and secondary kinetic isotope effects for the ring opening of the DHO were determined from analysis of the N and C of the carbamyl group after hydrolysis. In addition, the beta-carboxyl of the residual aspartate was liberated enzymatically by transamination to oxaloacetate with aspartate aminotransferase and then decarboxylation with oxaloacetate decarboxylase. The 13C/12C ratio from the released CO2 was determined by IRMS, yielding a second primary isotope effect. The primary and secondary isotope effects for the reaction catalyzed by DHOase showed little variation between enzymes or temperatures, the primary 13C and 15N isotope effects being approximately 1% on average, while the secondary 13C isotope effect is negligible or very slightly normal (>1.0000). These data indicate that the chemistry is at least partially rate-limiting while the secondary isotope effects suggest that the transition state may have lost some bending and torsional modes leading to a slight lessening of bond stiffness at the carbonyl carbon of the amide of CA-asp. The equilibrium isotope effects for DHO --> CA-asp have also been measured (secondary 13K(eq) = 1.0028 +/- 0.0002, primary 13K(eq) = 1.0053 +/- 0.0003, primary 15K(eq) = 1.0027 +/- 0.0003). Using these equilibrium isotope effects, the kinetic isotope effects for the physiological reaction (CA-asp --> DHO) have been calculated. These values indicate that the carbon of the amide group is more stiffly bonded in DHO while the slightly lesser, but still normal, values of the primary kinetic isotope effect show that the chemistry remains at least partially rate-limiting for the physiological reaction. It appears that the ring opening and closing is the slow step of the reaction.

Probing Nitrogen-Sensitive Steps in the Free-Radical-Mediated Deamination of Amino Alcohols by Ethanolamine Ammonia-Lyase

The contribution of C-N bond-breaking/making steps to the rate of the free-radical-mediated deamination of vicinal amino alcohols by adenosylcobalamin-dependent ethanolamine ammonia-lyase has been investigated by 15N isotope effects (IE's) and by electron paramagnetic resonance (EPR) spectroscopy. 15N IE's were determined for three substrates, ethanolamine, (R)-2-aminopropanol, and (S)-2-aminopropanol, using isotope ratio mass spectrometry analysis of the product ammonia. Measurements with all three substrates gave measurable, normal 15N IE's; however, the IE of (S)-2-aminopropanol was approximately 5-fold greater than that of the other two. Reaction mixtures frozen during the steady state show that the 2-aminopropanols give EPR spectra characteristic of the initial substrate radical, whereas ethanolamine gives spectra consistent with a product-related radical (Warncke, K.; Schmidt, J. C.; Kee, S.-C. J. Am. Chem. Soc. 1999, 121, 10522-10528). The steady-state concentration of the radical with (R)-2-aminopropanol is about half that observed with the S isomer, and with (R)-2-aminopropanol, the steady-state level of the radical is further reduced upon deuteration at C1. The results show that relative heights of kinetic barriers differ among the three substrates such that levels or identities of steady-state intermediates differ. 15N-sensitive steps are significant contributors to V/K with (S)-2-aminopropanol.

Metal ion catalyzed hydrolysis of ethyl p-nitrophenyl phosphate

15N isotope effects in the nitro group and 18O isotope effects in the phenolic oxygen have been measured for the hydrolysis of ethyl p-nitrophenyl phosphate catalyzed by several metal ions. Co(III)–cyclen at pH 7, 50 °C, gave an 15N isotope effect of 0.12% and an 18O one of 2.23%, showing that P–O cleavage is rate limiting and the bond is ∼50% broken in the transition state. The active catalyst is a dimer and the substrate is presumably coordinated to the open site of one Co(III), and is attacked by hydroxide coordinated to the other Co(III). Co(III)–tacn under the same conditions shows a similar 15N isotope effect (0.13%), but a smaller 18O one (0.8%). Zn(II)–cyclen at pH 8.5, 80 °C, gave an 15N isotope effect of 0.05% and an 18O one of 0.95%, suggesting an earlier transition state. The catalyst in this case is monomeric, and thus the substrate is coordinated to one position and attacked by a cis-coordinated hydroxide. Eu(III) at pH 6.5, 50 °C, shows a very large 15N isotope effect of 0.34% and a 1.6% 18O isotope effect. The large 15N isotope effect argues for a late transition state or Eu(III) interaction with the nitro group, and was also seen in Eu(III)-catalyzed hydrolysis of p-nitrophenyl phosphate [Bioorg. Chem. 28 (2000) 283].

15N kinetic isotope effects on uncatalyzed and enzymatic deamination of cytidine.

15N isotope effects and solvent deuterium isotope effects have been measured for the hydrolytic deamination of cytidine catalyzed by Escherichia coli cytidine deaminase and for the uncatalyzed reaction proceeding spontaneously in neutral solution at elevated temperatures. The primary (15)(V/K) arising from the exocyclic amino group for wild-type cytidine deaminase acting on its natural substrate, cytidine, is 1.0109 (in H(2)O, pH 7.3), 1.0123 (in H(2)O, pH 4.2), and 1.0086 (in D(2)O, pD 7.3). Increasing solvent D(2)O content has no substantial effect on k(cat) but enhances k(cat)/K(m), with a proton inventory showing that the fractionation factors of at least two protons increase markedly during the reaction. Mutant cytidine deaminases with reduced catalytic activity show more pronounced (15)N isotope effects of 1.0124 (Glu91Ala), 1.0134 (His102Ala), and 1.0158 (His102Asn) at pH 7.3 in H(2)O, as expected for processes in which the chemical transformation of the substrate becomes more rate determining. The isotope effect of mutant His102Asn is 1.033 after correcting for protonation of the -NH(2) group, and represents the intrinsic isotope effect on C-N bond cleavage. This result allows an estimation of the forward commitment of the reaction with the wild-type enzyme. The observed (15)N kinetic isotope effect of the pyrimidine N-3, for wild-type cytidine deaminase acting on cytidine, is 0.9879, which is consistent with protonation and rehybidization of N-3 with hydroxide ion attack on the adjacent carbon to create a tetrahedral intermediate. These results show that enzymatic deamination of cytidine proceeds stepwise through a tetrahedral intermediate with ammonia elimination as the major rate-determining step. The primary (15)N isotope effects observed for the uncatalyzed reaction at pH 7 (1.0021) and pH 12.5 (1.0034) were found to be insensitive to changing temperatures between 100 and 185 degrees C. These results show that the uncatalyzed and the enzymatic deaminations of cytidine proceed by similar mechanisms, although the commitment to C-N bond breaking is greater for the spontaneous reaction.

Kinetic Isotope Effects in the N2O Decomposition over NiO

The 15N and 18O kinetic isotope effects (KIEs) in the catalytic decomposition of nitrous oxide on NiO powder were determined in the temperature range of 625−825 K, and the following temperature dependencies were found: KIE(15N) = (0.821 ± 0.180) + (1445 ± 128)/T and KIE(18O) = (1.384 ± 0.124) + (1450 ± 89)/T. At initial pressures of N2O between 40 and 60 kPa, the reaction is first order in N2O and has an apparent activation energy of 131 ± 3 kJ mol-1. According to Bigeleisen's formalism, a bent, product-like NNO transition state and a fork-type NNOO transition state best fit the experimental results.

Lanthanide Catalyzed Cyclization of Uridine 3′-p-Nitrophenyl Phosphate

Steady state kinetics and 15N isotope effects have been used to study the cyclization reaction of uridine 3′-p-nitrophenyl phosphate. The cyclization reaction is catalyzed by transition metal ions and lanthanides, as are substitution reactions of many phosphate esters. Kinetic analysis reveals that the erbium-catalyzed cyclization reaction involves the concerted deprotonation of the 2′-OH group and departure of the leaving group. The transition state is very late, with a very large degree of bond cleavage to the leaving group, which could be due to a large degree of polarization of the PO bonds by erbium.

Kinetic Isotope Effects and Stereochemical Studies on a Ribonuclease Model: Hydrolysis Reactions of Uridine 3′-Nitrophenyl Phosphate

The reactions of a ribonuclease model substrate, the compound uridine-3′-p-nitrophenyl phosphate, have been examined using heavy-atom isotope effects and stereochemical analysis. The cyclization of this compound is subject to catalysis by general base (by imidazole buffer), specific base (by carbonate buffer), and by acid. All three reactions proceed by the same mechanistic sequence, via cyclization to cUMP, which is stable under basic conditions but which is rapidly hydrolyzed to a mixture of 2′- and 3′-UMP under acid conditions. The isotope effects indicate that the specific base-catalyzed reaction exhibits an earlier transition state with respect to bond cleavage to the leaving group compared to the general base-catalyzed reaction. Stereochemical analysis indicates that both of the base-catalyzed reactions proceed with the same stereochemical outcome. It is concluded that the difference in the nucleophile in the two base-catalyzed reactions results in a difference in the transition state structure but both reactions are most likely concerted, with no phosphorane intermediate. The 15N isotope effects were also measured for the reaction of the substrate with ribonuclease A. The results indicate that considerably less negative charge develops on the leaving group in the transition state than for the general base-catalyzed reaction in solution.

15N isotope effects in glutamine hydrolysis catalyzed by carbamyl phosphate synthetase: evidence for a tetrahedral intermediate in the mechanism.

15N isotope effects have been measured on the hydrolysis of glutamine catalyzed by carbamyl phosphate synthetase of Escherichia coli. The isotope effect in the amide nitrogen of glutamine is 1. 0217 at 37 degrees C with the wild-type enzyme in the presence of MgATP and HCO(3)(-) (overall reaction taking place). This V/K isotope effect indicates that breakdown of the tetrahedral intermediate formed with Cys 269 to release ammonia is the rate-limiting step in the hydrolysis. A full isotope effect of 1. 0215 is also seen in the partial reaction catalyzed by an E841K mutant enzyme, whose rate of glutamine hydrolysis is not affected by MgATP and HCO(3)(-). With wild-type enzyme in the absence of MgATP and HCO(3)(-), however, the (15)N isotope effect is reduced to 1. 0157. These isotope effects are interpreted in terms of partitioning of the tetrahedral intermediate whose rate of formation is dependent upon a conformation change which closes the active site after glutamine binding and prepares the enzyme for catalysis. An Ordered Uni Bi mechanism for glutamine hydrolysis that is consistent with the isotope effects and with the catalytic properties of the enzyme is proposed.

13C, 15N, and 18O equilibrium isotope effects and fractionation factors1

A listing of 13C, 15N, and 18O equilibrium isotope effects and fractionation factors for atoms in specific positions is provided. Empirical factors that can be used to adjust these fractionation factors for more complex structures are presented and discussed. While much work needs to be done to determine equilibrium isotope effects for these heavy atoms, the values tabulated here should be useful to anyone working with isotope effects involving these atoms.Key words: equilibrium isotope effects, fractionation factors, 13C, 15N, 18O.