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PubMed1994

Primary and secondary kinetic isotope effects as probes of the mechanism of yeast enolase.

Enolase catalyzes the interconversion of 2-phosphoglycerate and phosphoenolpyruvate. Kinetic isotope effects have been used to determine whether abstraction of the proton from C-2 and loss of hydroxide from C-3 of 2-phosphoglycerate occur in a concerted reaction or as sequential processes and whether these steps are kinetically significant for the enolase-catalyzed reaction. Enolase exhibits a significant primary deuterium isotope effect, as well as catalyzing the relatively rapid exchange of the C-2 proton with solvent water. Secondary C-3 deuterium isotope effects are also reported, both when the C-2 carbon carries a hydrogen and when this center is deuterated. These results provide information about the kinetic significance and timing of the transition state(s) associated with the loss of H+ and OH-. Strong evidence has been presented for a stepwise mechanism where both the rate of proton abstraction and one or both of the later transition states, i.e., those associated with hydroxide loss and product release, limit the overall reaction rate. If a concerted reaction were to be invoked, the presence of a small secondary 2H isotope effect in combination with the observed rate of exchange of the C-2 proton require the intrinsic secondary 2H kinetic isotope effect to be effectively unity. For the concerted mechanism, an intrinsic effect of unity would be consistent only with an extremely asymmetric transition state that is dominated by C-H bond cleavage.

Deuterium↗
PubMed1991

Multiple isotope effects with alternative dinucleotide substrates as a probe of the malic enzyme reaction.

Deuterium isotope effects and 13C isotope effects with deuterium- and protium-labeled malate have been obtained for both NAD- and NADP-malic enzymes by using a variety of alternative dinucleotide substrates. With nicotinamide-containing dinucleotides as the oxidizing substrate, the 13C effect decreases when deuterated malate is the substrate compared to the value obtained with protium-labeled malate. These data are consistent with a stepwise chemical mechanism in which hydride transfer precedes decarboxylation of the oxalacetate intermediate as previously proposed [Hermes, J. D., Roeske, C. A., O'Leary, M. H., & Cleland, W. W. (1982) Biochemistry 21, 5106]. When dinucleotide substrates such as thio-NAD, 3-acetylpyridine adenine dinucleotide, and 3-pyridinealdehyde adenine dinucleotide that contain modified nicotinamide rings are used, the 13C effect increases when deuterated malate is the substrate compared to the value obtained with protium-labeled malate. These data, at face value, are consistent with a change in mechanism from stepwise to concerted for the oxidative decarboxylation portion of the mechanism. However, the increase in the deuterium isotope effect from 1.5 to 3 with a concomitant decrease in the 13C isotope effect from 1.034 to 1.003 as the dinucleotide substrate is changed suggests that the reaction may still be stepwise with the non-nicotinamide dinucleotides. A more likely explanation is that a beta-secondary 13C isotope effect accompanies hydride transfer as a result of hyperconjugation of the beta-carboxyl of malate as the transition state for the hydride transfer step is approached.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Aspartate beta-decarboxylase from Alcaligenes faecalis: carbon-13 kinetic isotope effect and deuterium exchange experiments.

We have measured the 13C kinetic isotope effect at pH 4.0, 5.0, 6.0, and 6.5 and in D2O at pD 5.0 and the rate of D-H exchange of the alpha and beta protons of aspartic acid in D2O at pD 5.0 for the reaction catalyzed by the enzyme aspartate beta-decarboxylase from Alcaligenes faecalis. The 13C kinetic isotope effect, with a value of 1.0099 +/- 0.0002 at pH 5.0, is less than the intrinsic isotope effect for the decarboxylation step, indicating that the decarboxylation step is not entirely rate limiting. We have been able to estimate probable values of the relative free energies of the transition states of the enzymatic reaction up to and including the decarboxylation step from the 13C kinetic isotope effect and the rate of D-H exchange of alpha-H. The pH dependence of the kinetic isotope effect reflects the pKa of the pyridine nitrogen of the coenzyme pyridoxal 5'-phosphate but not that of the imine nitrogen. A mechanism is proposed for the exchange of aspartate beta-H that is consistent with the stereochemistry suggested earlier.

Alcaligenes↗
PubMed1987

Remote nitrogen-15 isotope effects on addition of cyanide to NAD.

The reversible reaction NAD + CN(-)----NAD-CN was examined for remote secondary 15N isotope effects caused by isotopic substitution at the ring nitrogen of the nicotinamide group. These were compared with analogous effects for dehydrogenase-catalyzed reactions, since both cyanide and the hydride ion add at the N-4 position of the nicotinamide ring. The 15N effects on the rate constants for the forward and reverse processes were examined directly by conducting both the normal and isotopic reactions simultaneously under carefully controlled conditions in the sample and reference cells of a dual-beam spectrophotometer. In both cases, the 15N kinetic isotope effect differed from 1.00 by considerably less than 0.01. The 15N equilibrium isotope effect, 15K, was obtained as the ratio of equilibrium constants measured separately with natural-abundance and labeled NAD by using a concentration jump procedure [1.004 +/- 0.002 (cyanide addition)]. A similar value for 15K of 1.010 +/- 0.008 was obtained in an analogous manner for the reaction catalyzed by lactate dehydrogenase: NAD + lactate----pyruvate + NADH + H+. The latter value is significantly smaller than a previously reported value obtained from kinetic studies [1.044 +/- 0.012; Cook, P. F., Oppenheimer, N. J., & Cleland, W. W. (1981) Biochemistry 20, 1817]. The present value also is smaller than might be expected for a change in bond order from 4 to 3 [Cleland, W. W. (1980) Methods Enzymol. 64, 104-125] on the basis of the canonical resonance structures for NAD and NADH.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Primary deuterium isotope effects for the 3-methylaspartase-catalyzed deamination of (2S)-aspartic acid, (2S,3S)-3-methylaspartic acid, and (2S,3S)-3-ethylaspartic acid.

3-Methylaspartate ammonia-lyase catalyzes the deamination of (2S)-aspartic acid 137 times more slowly than the deamination of (2S,3S)-3-methylaspartic acid but catalyzes the amination of fumaric acid 1.8 times faster than the amination of mesaconic acid [Botting, N.P., Akhtar, M., Cohen, M. A., & Gani, D. (1988) Biochemistry (preceding paper in this issue)]. In order to understand the mechanistic basis for these observations, the deamination reaction was examined kinetically with (2S)-aspartic acid, (2S,3S)-3-methylaspartic acid, (2S,3S)-3-ethylaspartic acid, and the corresponding C-3-deuteriated isotopomers. Comparison of the double-reciprocal plots of the initial reaction velocities for each of the three pairs of substrates revealed that the magnitude of the primary isotope effect on both Vmax and V/K varied with the substituent at C-3 of the substrate. 3-Methylaspartic acid showed the largest isotope effect (1.7 on Vmax and V/K), 3-ethylaspartic acid showed a smaller isotope effect (1.2 on Vmax and V/K), and aspartic acid showed no primary isotope effect at all. These results, which are inconsistent with earlier reports that there is no primary isotope effect for 3-methylaspartic acid [Bright, H. J. (1964) J. Biol. Chem. 239, 2307], suggest that for both 3-methylaspartic acid and 3-ethylaspartic acid elimination occurs via a predominantly concerted mechanism whereas for aspartic acid an E1cb mechanism prevails.(ABSTRACT TRUNCATED AT 250 WORDS)

Ammonia-Lyases↗
PubMed2001

Tritium partitioning and isotope effects in adenosylcobalamin-dependent glutamate mutase.

Tritiated adenosylcobalamin, labeled at the exchangeable position, has been used to investigate the partitioning of tritium between substrate and product in the reaction catalyzed by glutamate mutase. The isotope partitions between glutamate and methylaspartate in nearly 1:1 ratio, regardless of the direction in which the overall reaction is proceeding. This is consistent with a free-energy profile in which the interconversion of the intermediate glutamyl and methylaspartyl radicals is rapid relative to the transfer of tritium from 5'-deoxyadenosine to either substrate or product. Initial velocity measurements have been used to measure the tritium isotope effects for the transfer of tritium from adenosylcobalamin to product in each direction. The isotope effect is 21 for the formation of glutamate and 19 for the formation of methylasparate. The large magnitude of these isotope effects makes it likely that the rate-determining step may be altered by the substitution of tritium for hydrogen in the reaction. The results of these experiments are compared with previous isotope effect measurements made on other adenosylcobalamin-dependent enzymes.

Chromatography, High Pressure Liquid↗
PubMed2006

Mechanism of O2 activation by cytochrome P450cam studied by isotope effects and transient state kinetics.

The early steps in dioxygen activation by the monooxygenase cytochrome P450cam (CYP101) include binding of O2 to ferrous P450cam to yield the ferric-superoxo form (oxyP450cam) followed by an irreversible, long-range electron transfer from putidaredoxin to reduce the oxyP450cam. The steady state kinetic parameter kcat/Km(O2) has been studied by a variety of probes that indicate a small D2O solvent isotope effect (1.21 +/- 0.08), a very small solvent viscosogen effect, and a 16O/18O isotope effect of 1.0147 +/- 0.0007. This latter value, which can be compared with the 16O/18O equilibrium isotope effect of 1.0048 +/- 0.0003 measured for oxyP450cam formation, is attributed to a primarily rate-limiting outer-sphere electron transfer from the heme iron center as O2 that has prebound to protein approaches the active site cofactor. The electron transfer from putidaredoxin to oxyP450cam was investigated by rapid mixing at 25 degrees C to complement previous lower-temperature measurements. A rate of 390 +/- 23 s-1 (and a near-unity solvent isotope effect) supports the view that the long-range electron transfer from reduced putidaredoxin to oxyP450cam is rapid relative to dissociation of O2 from the enzyme. P450cam represents the first enzymatic reaction of O2 in which both equilibrium and kinetic 16O/18O isotope effects have been measured.

Bacterial Proteins↗
PubMed2006

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.

Alanine↗
PubMed1997

Isotope effect studies on the calcineurin phosphoryl-transfer reaction: transition state structure and effect of calmodulin and Mn2+.

The hydrolysis of p-nitrophenyl phosphate (pNPP) catalyzed by calcineurin has been studied by measurement of heavy-atom isotope effects in the substrate. The isotope effects were measured at the nonbridging oxygen atoms [18(V/K)nonbridge], at the position of bond cleavage in the bridging oxygen atom [18(V/K)bridge], and at the nitrogen atom in the nitrophenol leaving group [15(V/K)]. The isotope effects increased in magnitude upon moving from the pH optimum of 7.0 to 8.5; 18(V/K)bridge increased from 1.0072 to 1.0115, and 15(V/K) from 1.0006 to 1.0014. The value for 18(V/K)nonbridge is 0.9942 at pH 8.5. These data are consistent with P-O bond cleavage being partially rate-limiting at the pH optimum and more so at the higher pH. The 18(V/K)nonbridge isotope effect indicates that the dianion is the substrate for catalysis, and a dissociative transition state is operative for phosphoryl transfer. Increasing the concentration of the activating metal ion Mn2+ at pH 7.0 from 1 mM to 5 mM increases the magnitude of the isotope effects by an amount similar to that observed with the shift in pH from 7.0 to 8.5, indicative of a change in the commitment factor in the kinetic mechanism so as to make the chemical step more rate-limiting.

Animals↗
PubMed2001

Cadmium reactivity in metal-contaminated soils using a coupled stable isotope dilution-sequential extraction procedure.

To better understand the intrinsic reactivity of Cd, four soils having diverse sources of Cd contamination (total Cd: 22-34 mg kg(-1)) were investigated using a stable isotope dilution-sequential extraction procedure (SID-SEP) during a 59-week incubation. Samples were spiked with carrier-free 111Cd and periodically extracted into five operationally defined fractions; the 111Cd:10Cd ratios were measured by inductively coupled plasma-mass spectrometry. The total labile pool of Cd (E value) was calculated as well as the labile Cd within each extracted fraction. Results for three of the soils (Cd sources: natural, sewage sludge, smelter emissions) were quite similar. The overall %E after 2-week equilibration was 35-49% of total soil Cd. Within fraction 2 (sorbed/carbonate), 70-75% of the Cd was isotopically labile, while within fraction 3 (oxidizable) only 35-41% of the Cd was labile within 2 weeks. The fourth (reducible) and fifth (residual) fractions were dominated by nonlabile Cd. Although all E values increased somewhat from 2 to 59 weeks, none of the extracted fractions reached isotopic equilibrium with the soluble/exchangeable Cd extracted during step 1. Because fractions 2 and 3 dominated the native Cd in all three soils, the total labile pool was contributed primarily (85-98%) by these two fractions. A fourth soil (mine spoil-contaminated) was demonstrably different: after 2 weeks, the overall %E was just 13 and, although 82% of the total Cd was present in the oxidizable fraction, just 2% of that was isotopically labile. The nonlability of Cd in this soil could be ascribed to the predominance of inorganic forms, most likely occluded Cd in sphalerite. No single Cd fraction from the SEP nor any combination of fractions showed a good correspondence with the size of the isotopically labile pool. Our results suggest that conventional SEPs may be of limited utility for predicting bioavailability, for example, during ecological risk assessment.

Biological Availability↗
PubMed2002

Molecular and stable carbon isotopic source identification of oil residues and oiled bird feathers sampled along the Atlantic Coast of France after the Erika oil spill.

The Erika tanker broke in two close to the Atlantic coast of France on December 12, 1999. On December 25th, some heavy fuel oil released by the tanker came ashore along the French Atlantic Coast. Some oil residues and oiled bird feathers were collected all along the Atlantic Shoreline of France after the wreck of the Erika tanker. The aim of this study was to differentiate oil residues and oiled bird feathers related to the Erika oil spill from the ones resulting from the numerous tar ball incidents which had occurred after the Erika oil spill. Alkane and PAH quantification of oil residues allowed differentiation of the samples collected on the north part of the Atlantic Coast from those collected on the south part of the Atlantic shoreline. All oiled birds appear to have been contaminated by the Erika oil. Samples collected on the south part of the Atlantic Coast contain a different molecular fingerprint compared to the Erika oil indicating that they are not related to the Erika oil spill. Bulk and molecular 13C/12C ratio measurements were performed in order to check the discriminative feature and the stability of the isotopic approach. Bulk stable carbon isotopic composition has been shown to be a valuable screening correlation tool as it confirms the link of samples collected in the north part of the Atlantic Coast with the Erika oil spill. All the samples collected along the south part of the Atlantic Shoreline exhibit 13C-enriched bulk isotopic compositions compared to Erika oil. Molecular isotopic composition of saturated hydrocarbons and of phenanthrene compounds also allows unambiguous differentiation of samples related to the Erika oil spill from those due to tar ball incidents. Over the long-term, when molecular distribution will have been modified by the different processes affecting oil in the marine environment, molecular isotopic composition should then be of particular help for Erika oil residues identification.

Animals↗
PubMed2004

Historical variations in the stable isotope composition of mercury in Arctic lake sediments.

The stable isotope composition of mercury (Hg) in a dated core from the anoxic zone of a saline, meromictic Arctic lake was found to vary as a complex function of the age and chemical composition of the sediment. Throughout the stratigraphic sequence, which spans the years 1899-1997, the ratios 198Hg/202Hg, 199Hg/202Hg, 200Hg/202Hg, 201Hg/202Hg, and 204Hg/202Hg expressed as delta-values (per mil deviations relative to a standard) reveal enrichment in 198Hg, 199Hg, 200Hg, and 201Hg, with depletion in 204Hg, the degree of enrichment varying inversely with atomic mass. A plot of delta198Hg, delta199Hg, delta200Hg, and delta201Hg against depth gave parallel profiles characterized by large, regular undulations superimposed on an overall trend toward increase with depth (i.e. age), and the delta204Hg profile is a mirror image of them. The delta198Hg, delta199Hg, delta200Hg, and delta201Hg values of the oldest (1899-1929) strata vary inversely with NH2OH.HCl/HNO3-extractable manganese concentration, but those of the youngest (1963-1997) strata give a positive correlation; intermediate (1936-1956) strata show no correlation and negligible variation in delta-values, possibly signifying a transition phase in which the two opposite trends offset each other. The delta-values show similar but weaker relationships with organic carbon. The results strongly suggest fractionation of Hg isotopes by microbial activities linked to oxidation-reduction reactions in the lake, although effects of isotopic signatures indicative of the sources of the Hg have not been ruled out. The radical change in the nature of the relationship between 6-values and sediment chemistry over time may reflect environmental and biotic changes that altered the isotope-fractionating processes. These findings imply that variations in the isotopic makeup of Hg, together with related physical, chemical, and biological data, could yield important new information about the biogeochemical cycle of Hg.

Arctic Regions↗
PubMed2004

Hydrogen and carbon isotope fractionation during anaerobic biodegradation of aromatic hydrocarbons--a field study.

The aquifer of a former manufactured gas plant site, highly contaminated by dissolved monocyclic, heterocyclic, and polycyclic aromatic hydrocarbons, was studied to evaluate the applicability of carbon and hydrogen isotope fractionation to prove ongoing biodegradation of these compounds even in complex aquifer settings. The loss of toluene, o-xylene, p,m-xylene, and 2-methylnaphthalene was accompanied by a considerable carbon isotope fractionation. Additionally, a strong 2H enrichment in residual o-xylene was detected. All isotope fractionations observed could be related to established biochemical degradation mechanisms, each involving a C-H bond cleavage in the rate-determining step. In contrast, other compounds such as 1-methylnaphthalene, methylbenzofuran, and acenaphthene exhibited a uniform stable carbon isotope composition. However, a decrease in concentration for these compounds was observed in the flowpath of the aquifer. High threshold concentrations of acenaphthene downgradient indicate that this contaminant is, if at all, only marginally biodegraded. Detailed analyses of xylenes provided support that compound specific isotope analyses and subsequent application of the Rayleigh model may provide a valuable basis to distinguish between different biodegradation mechanisms as well as dissolution processes in heterogeneous aquifers.

Bacteria, Anaerobic↗
PubMed2005

Carbon isotopic fractionation during anaerobic biotransformation of methyl tert-butyl ether and tert-amyl methyl ether.

The fuel oxygenate methyl tert-butyl ether (MTBE) has been frequently detected in groundwater and surface water. Since contaminated sites are often subsurface, anaerobic degradation of MTBE will likely be significant for remediation. As traditional approaches to evaluate biodegradation generally involve laboratory microcosm studies which require time and resources, innovative approaches are needed to demonstrate active in situ biodegradation of MTBE. This study was conducted to gather information at the laboratory level to evaluate the potential of applying carbon isotope fractionation as an indicator for in situ biodegradation of the fuel oxygenates MTBE and tert-amyl methyl ether (TAME). In this study, MTBE utilization was observed in a methanogenic sediment microcosm after a lengthy lag period of about 400 days. MTBE utilization was sustained upon refeeding and subculturing. tert-Butyl alcohol (TBA) was found to accumulate after propagation of cultures. The MTBE-grown cultures also utilized TAME and produced tert-amyl alcohol (TAA). The detection of TBA and TAA indicated that ether bond cleavage was the initial step in degradation for both compounds. Carbon isotope fractionation during anaerobic MTBE and TAME degradation was studied, and isotopic enrichment factors (epsilon) with 95% confidence intervals of -15.6 +/-4.1% and -13.7+/-4.5% were estimated for anaerobic MTBE and TAME degradation, respectively. Addition of 2-bromoethanesulfonic acid, an inhibitor of methanogenesis, substantially prolonged the lag period before transformation, but did not influence carbon isotope fractionation. Our experiment provided strong evidence of significant carbon isotope fractionation during anaerobic MTBE and TAME degradation, demonstrating that this technique can be used as an indicator for in situ MTBE and TAME degradation.

Air Pollutants↗
PubMed2005

Assessing the potential for re-emission of mercury deposited in precipitation from arid soils using a stable isotope.

A solution containing 198Hg in the form of HgCl2 was added to a 4 m2 area of desert soils in Nevada, and soil Hg fluxes were measured using three dynamic flux chambers. There was an immediate release of 198Hg after it was applied, and then emissions decreased exponentially. Within the first 6 h after the isotope was added to the soil, approximately 12 ng m(-2) of 198Hg was emitted to the atmosphere, followed by a relatively steady flux of the isotope at 0.2 +/- 0.2 ng m(-2) h(-1) for the remainder of the experiment (62 days). Over this time, approximately 200 ng m(-2) or 2% of the 198Hg isotope was emitted from the soil, and we estimate that approximately 6% of the isotope would be re-emitted in a year's time. During the experiment, dry deposition of elemental Hg from the atmosphere was measured with an average deposition rate of 0.2 +/- 0.1 ng m(-2) h(-1). Emission of ambient Hg from the soil was observed after soil wetting with the isotope solution and after a storm event. However, the added moisture from the storm event did not affect 198Hg flux. Results suggest that in this desert environment, where there is limited precipitation, Hg deposited by wet processes is not readily re-emitted and that dry deposition of elemental Hg may be an important process.

Atmosphere↗
PubMed2006

Does the Rayleigh equation apply to evaluate field isotope data in contaminant hydrogeology?

Stable isotope data have been increasingly used to assess in situ biodegradation of organic contaminants in groundwater. The data are usually evaluated using the Rayleigh equation to evaluate whether isotope data follow a Rayleigh trend, to calculate the extent of contaminant biodegradation, or to estimate first-order rate constants. However, the Rayleigh equation was developed for homogeneous systems while in the subsurface, contaminants can migrate at different velocities due to physical heterogeneity. This paper presents a method to quantify the systematic effect that is introduced by applying the Rayleigh equation to field isotope data. For this purpose, the travel time distribution between source and sampling point is characterized by an analytical solution to the advection-dispersion equation. The systematic effect was evaluated as a function of the magnitude of physical heterogeneity, geometry of the contaminant plume, and degree of biodegradation. Results revealed that the systematic effect always leads to an underestimation of the actual values of isotope enrichment factors, the extent of biodegradation, or first-order rate constants, especially in the dispersion-dominant region representing a higher degree of physical heterogeneity. A substantial systematic effect occurs especially for the quantification of first-order rate constants (up to 50% underestimation of actual rate) while it is relatively small for quantification of the extent of biodegradation (< 5% underestimation of actual degree of biodegradation). The magnitude of the systematic effect is in the same range as the uncertainty due to uncertainty of the analytical data, of the isotope enrichment factor, and the average travel time.

Biodegradation, Environmental↗
PubMed2006

Evaluation of isotopic enrichment factors for the biodegradation of chlorinated ethenes using a parameter estimation model: toward an improved quantification of biodegradation.

A model was developed to predict the concentrations of chlorinated ethenes and ethene during sequential reductive dechlorination of tetrachloroethene (PCE) from stable carbon isotope values using Rayleigh model principles and specified isotopic enrichment factors for each step of dechlorination. The model was tested using three separate datasets of concentration and isotope values measured during three experiments involving the degradation of PCE to vinyl chloride (VC), trichloroethene (TCE) to ethene, and cis-1,2-dichloroethene (cDCE) to ethene. The model was then coupled to a parameter estimation method to estimate values for the isotopic enrichment factors of TCE, cDCE, and VC when they are intermediates in the dechlorination to ethene. The enrichment factors estimated for TCE and cDCE when they were intermediates in biodegradation experiments were close to or within the published range of enrichment factors determined from experiments where TCE or cDCE were the initial substrates. In contrast, the enrichment factors determined by parameter estimation for experiments in which VC was an intermediate in biodegradation experiments were consistently more negative (by approximately 10 per thousandth) than the most negative published enrichment factor determined from experiments where VC was the initial substrate. This finding suggests that the range of enrichment factors for VC dechlorination may not be as narrow as previously suggested (-21.5 per thousandth to -26.6 per thousandth) and that fractionation during VC dechlorination when VC is an intermediate compound may be significantly larger than when VC is the initial substrate. These findings have important implications both for the current practice of extrapolating laboratory-derived isotopic enrichment factors to quantify biodegradation of chlorinated ethenes in the field and for understanding the details of enzymatic reductive dechlorination.

Biodegradation, Environmental↗
PubMed2001

Theoretical studies of mechanisms and kinetic isotope effects on the decarboxylation of orotic acid and derivatives.

The mechanism of orotidine 5'-monophosphate decarboxylase was studied computationally by using the decarboxylation of orotic acid analogues as model systems. These calculations indicate that mechanisms involving proton transfer to the 2-oxygen or the 4-oxygen are energetically favorable, as compared to direct decarboxylation without proton transfer, for a series of model compounds where N1 is substituted with respectively H, CH(3), and a tetrahydrofuran moiety. Proton transfer to the 4-oxygen during decarboxylation is found to be energetically more favorable than 2-protonation, which is attributable to both the 4-oxygen site being more basic and an apparent intrinsic preference for the 4-protonation pathway. (15)N isotope effect calculations were also conducted, and compared to experimental (15)N isotope effects previously measured at N1 by Rishavy and Cleland (Biochemistry 2000, 39, 4569-4574). The theoretical isotope effects establish, for the first time, that the experimental (15)N isotope effect is consistent with decarboxylation without protonation, as well as with decarboxylation with protonation, at either O2 or at O4. Furthermore, we propose herein an isotope measurement that could potentially distinguish among mechanisms involving protonation from those that do not involve proton transfer.

Kinetics↗