Human erythrocyte glutathione reductase: chemical mechanism and structure of the transition state for hydride transfer

Kinetic parameters and primary deuterium kinetic isotope effects for NADH and five pyridine nucleotide substrates have been determined at pH 8.1 for human erythrocyte glutathione reductase. DV/KNADH and DV are equal to 1.4 and are pH independent below pH 8.1, but DV decreases to 1.0 at high pH as a group exhibiting a pK of 8.6 is deprotonated. This result suggests that as His-467' is deprotonated, the rate of the isotopically insensitive oxidative half-reaction is specifically decreased and becomes rate-limiting. For all substrates, equivalent V and V/K primary deuterium kinetic isotope effects are observed at pH values below 8.1. The primary deuterium kinetic isotope effect on V, but not V/K, is sensitive to solvent isotopic composition. The primary tritium kinetic isotope effects agree well with the corresponding value calculated from the primary deuterium kinetic isotope effects by using the Swain-Schaad relationship. This suggests that the primary deuterium kinetic isotope effects observed in these steady-state experiments are the intrinsic primary deuterium kinetic isotope effects for hydride transfer. The magnitude of the primary deuterium kinetic isotope effect is dependent on the redox potential of the pyridine nucleotide substrate used, varying from approximately 1.4 for NADH and -320 mV reductants to 2.7 for thioNADH to 4.2-4.8 for 3-acetylpyridine adenine dinucleotide (3APADH). The alpha-secondary tritium kinetic isotope effects also increase as the redox potential of the pyridine nucleotide substrate becomes more positive. Together, these data indicate that the transition state for hydride transfer is very early for NADH and becomes later for thioNADH and 3APADH, as predicted by Hammond's postulate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetic isotope effects on cytochrome P-450-catalyzed oxidation reactions. The oxidative O-dealkylation of 7-ethoxycoumarin

The primary deuterium and tritium isotope effects on Vm/Km and on Vm have been measured for the O-deethylation of 7-ethoxycoumarin catalyzed by two purified isozymes of cytochrome P-450. From these data the intrinsic isotope effects have been calculated as described by D. B. Northrop (Biochemistry (1975) 14, 2644-2651). The observed deuterium isotope effects on Vm/Km are 3.79 and 1.90 for the isozymes isolated from the livers of rats induced by phenobarbital and 3-methylcholanthrene, respectively. The calculated intrinsic isotope effects, however, are similar and much larger (kH/kD = 12.8 to 14.0) than the observed isotope effects on Vm/Km for the two enzymes. This demonstrates that the intrinsic isotope effects are attenuated by various steps preceding the isotopically sensitive C-H bond cleavage step resulting in the low values for the observed isotope effects. Thus, the observed isotope effects do not accurately reflect the magnitude of the intrinsic isotope effect associated with this reaction. No incorporation of 18O into the 7-hydroxycoumarin product was observed in studies employing H218O or 18O2 demonstrating that the phenolic oxygen arises exclusively from the substrate. Taken together, these data provide compelling evidence that both cytochrome P-450 isozymes catalyze the O-dealkylation of this substrate by an identical radical recombination mechanism during the obligatory formation of a hemiacetal intermediate.     

Use of isotope effects to elucidate enzyme mechanisms

The chemical bond breaking steps are normally not rate limiting for enzymatic reactions. However, comparison of deuterium and tritium isotope effects on the same reaction, especially when coupled with 13C isotope effects for the same step measured with deuterated as well as unlabeled substrates, allows calculation of the intrinsic isotope effects on the bond breaking steps and thus a determination of the commitments to catalysis for the reactants. The variation in observed isotope effects as a function of reactant concentration can be used to determine kinetic mechanisms, while the pH variation of isotope effects can determine the stickiness of the reactants and which portions of the reactant mechanism are pH dependent. Finally the size of primary and secondary intrinsic isotope effects can be used to determine transition state structure.     

Deuterium isotope effects in the oxidation of alcohols in vitro and in vivo

The oxidation of ethanol and isopropanol by liver alcohol dehydrogenase was studied in vitro and in vivo. Oxidation of ethanol by horse liver alcohol dehydrogenase was carried out in the presence of lactaldehyde and other aldehydes which reoxidized enzyme-bound NADH. Under these conditions the oxidation of ethanol was accelerated 7 to 22-fold, depending on the nature of the aldehyde. (An acceleration of ethanol oxidation by lactaldehyde was previously reported by Gupta and Robinson [(1966) Biochim, Biophys. Acta118, 431]. In the presence of lactaldehyde ping-pong kinetics were observed and a deuterium isotope effect on V of 4.2 was seen. In the absence of acceptor aldehyde no, or small, isotope effects (Baker, R. H. (1962) Biochemistry1, 41) are observed. Therefore, when dissociation of NADH is no longer rate limiting the hydrogen transfer step becomes largely rate determining. Oxidation of isopropanol shows an isotope effect on V of 2.5 in the absence of acceptor aldehyde. With mouse liver alcohol dehydrogenase results similar to those obtained with the horse liver enzyme were obtained.When ethanol metabolism was examined in vivo, in mice by measuring blood alcohol levels, no isotope effect was observed with ethanol-1-d₂. On the other hand, an isotope effect of 2.0 was observed when the metabolism of isopropanol and isopropanol-2-d₁ were compared. This isotope effect is very close to that observed in vitro with the mouse liver enzyme. The relative rate of metabolism of ethanol and isopropanol in vivo was similar to that observed in vitro with the mouse liver enzyme (ethanol:isopropanol, 2.1 in vivo:2.2 in vitro). It was concluded that in the metabolism of ethanol and isopropanol, alcohol dehydrogenase is partially rate determining. Administration to mice of lactaldehyde, as well as other aldehydes, ketones, or fructose, simultaneously with ethanol produced no increase in the rate of ethanol metabolism.     

The use of isotope effects to determine enzyme mechanisms

Isotope effects are one of the most powerful kinetic tools for determining enzyme mechanisms. There are three methods of measurement. First, one can compare reciprocal plots with labeled and unlabeled substrates. The ratio of the slopes is the isotope effect on V/K, and the ratio of the vertical intercepts is the isotope effect on V(max). This is the only way to determine V(max) isotope effects, but is limited to isotope effects of 5% or greater. The second method is internal competition, where the labeled and unlabeled substrates are present at the same time and the change in their ratio in residual substrate or in product is used to calculate an isotope effect, which is that on V/K of the labeled reactant. This is the method used for tritium or (14)C, or with the natural abundances of (13)C, (15)N, or (18)O. The third method involves perturbations from equilibrium when a labeled substrate and corresponding unlabeled product are present at chemical equilibrium. This also gives just an isotope effect on V/K for the labeled reactant. The chemistry is typically not fully rate limiting, so that the isotope effect on V/K is given by: (x)(V/K)=((x)k+c(f)+c(r)(x)K(eq))/(1+c(f)+c(r)) where x defines the isotope (D, T, 13, 15, 18 for deuterium, tritium, (13)C, (15)N, or (18)O), and (x)(V/K), (x)k, and (x)K(eq) are the observed isotope effect, the intrinsic one on the chemical step, and the isotope effect on the equilibrium constant, respectively. The constants c(f) and c(r) are commitments in forward and reverse directions, and are the ratio of the rate constant for the chemical reaction and the net rate constant for release from the enzyme of the varied substrate (direct comparison) or labeled substrate (internal competition and equilibrium perturbation) for c(f), or the first product released or the one involved in the perturbation for c(r). The intrinsic isotope effect, (x)k, can be estimated by comparing deuterium and tritium isotope effects on V/K, or by comparing the deuterium isotope effect with (13)C ones with deuterated and undeuterated substrates. Adding a secondary deuterium isotope effect and its effect on the (13)C one can give an exact solution for all intrinsic isotope effects and commitments. The effect of deuteration on a (13)C isotope effect allows one to tell if the two isotope effects are on the same or different steps. Applications of these methods to several enzyme systems will be presented.     

Deuterium isotope effects in carbohydrates revisited. Cryoprobe studies of the anomerization and NH to ND deuterium isotope induced 13C NMR chemical shifts of acetamidodeoxy and aminodeoxy sugars

Complete ¹H and ¹³C NMR chemical shift assignments have been generated from a series of acetamidodeoxy and aminodeoxy sugar derivatives. For free sugars, the enhanced sensitivity of an NMR cryoprobe allowed simple 1D and 2D NMR spectra to be obtained from essentially single anomers, before significant mutarotation had occurred. The NMR assignments have been used to characterize deuterium isotope effects on ¹³C chemical shifts measured under conditions of slow NH to ND exchange in single solutions. Within a range of 0 to −0.138 ppm, β, γ, δ, and ζ deuterium isotope effects have been observed, thus providing additional reference data for assignment of the ¹³C NMR spectra of nitrogenous saccharides.     

Measurements and monitoring of the hydrogen and deuterium contents in the plasma of the L-2M stellarator

The program of experiments on ITER includes a sequential change of the plasma isotopic composition from pure hydrogen plasma in the initial stage of research to deuterium and, then, deuterium-tritium plasma with a gradual increase in the tritium content. In this context, the influence of the plasma isotopic composition on the processes of plasma heating and confinement are being actively studied on the existing tokamaks and stellarators. The plasma isotopic composition also depends on the composition of the gas desorbed from the vacuum chamber wall in the course of recycling. Therefore, the rate of change of the plasma isotopic composition after altering the injected gas also depends on the rate of change of the isotopic composition of the gas absorbed in the wall. These effects were studied in the experiments carried out on the L-2M stellarator in which the working gas was changed from hydrogen to deuterium. Spectral measurements of the intensity ratio between the H _α and D _α lines made it possible to monitor the isotopic composition of the plasma in the course of cleaning of the chamber wall from earlier absorbed hydrogen and its replacement with deuterium. After returning to hydrogen, the rate of cleaning of the wall from deuterium was also determined. The results of these experiments show that the plasma isotopic composition varies exponentially with the number N of shots after transition to another isotope, ∼exp(−N/47). Hence, the isotopic composition can be changed almost completely over 2 to 3 working days. This allows one to study the influence of the plasma isotopic composition on plasma confinement during the same experimental session.     

Carbon-13 and deuterium isotope effects on the reaction catalyzed by glyceraldehyde-3-phosphate dehydrogenase

Carbon-13 and deuterium isotope effects have been measured on the reaction catalyzed by rabbit muscle glyceraldehyde-3-phosphate dehydrogenase in an effort to locate the rate-limiting steps. With D-glyceraldehyde 3-phosphate as substrate, hydride transfer is a major, but not the only, slow step prior to release of the first product, and the intrinsic primary deuterium and 13C isotope effects on this step are 5-5.5 and 1.034-1.040, and the sum of the commitments to catalysis is approximately 3. The 13C isotope effects on thiohemiacetal formation and thioester phosphorolysis are 1.005 or less. The intrinsic alpha-secondary deuterium isotope effect at C-4 of the nicotinamide ring of NAD is approximately 1.4; this large normal value (the equilibrium isotope effect is 0.89) shows tight coupling of hydrogen motions in the transition state accompanied by tunneling. With D-glyceraldehyde as substrate, the isotope effects are similar, but the sum of commitments is approximately 1.5, so that hydride transfer is more, but still not solely, rate limiting for this slow substrate. The observed 13C and deuterium equilibrium isotope effects on the overall reaction from the hydrated aldehyde are 0.995 and 1.145, while the 13C equilibrium isotope effect for conversion of a thiohemiacetal to a thioester is 0.994, and that for conversion of a thioester to an acyl phosphate is 0.997. Somewhat uncertain values for the 13C equilibrium isotope effects on aldehyde dehydration and formation of a thiohemiacetal are 1.003 and 1.004.     

Kinetic mechanism and nucleotide specificity of NADH peroxidase

NADH peroxidase is a flavoprotein isolated from Streptococcus faecalis which catalyzes the pyridine nucleotide-dependent reduction of hydrogen peroxide to water. Initial velocity, product, and dead-end inhibition studies have been performed at pH 7.5 and support a ping-pong kinetic mechanism. In the absence of hydrogen peroxide, both transhydrogenation between NADH and thioNAD, and isotope exchange between [14C]NADH and NAD, have been demonstrated, although in both these experiments, the maximal velocity of nucleotide exchange was less than 1.5% the maximal velocity of the peroxidatic reaction. We propose that NADH binds tightly to both oxidized and two-electron reduced enzyme. NADH oxidation proceeds stereospecifically with the transfer of the 4S hydrogen to enzyme, and then, via exchange, to water. No primary tritium kinetic isotope effect was observed, and no statistically significant primary deuterium kinetic isotope effects on V/K were determined, although primary deuterium kinetic isotope effects on V were observed in the presence and absence of sodium acetate. NADH peroxidase thus shares with other flavoprotein reductases striking kinetic, spectroscopic, and stereochemical similarities. On this basis, we propose a chemical mechanism for the peroxide cleaving reaction catalyzed by NADH peroxidase which involves the obligate formation of a flavinperoxide, and peroxo bond cleavage by nucleophilic attack by enzymatic dithiols.     

Use of multiple isotope effects to study the mechanism of 6-phosphogluconate dehydrogenase

The multiple isotope effect method of Hermes et al. [Hermes, J. D., Roeske, C. A., O'Leary, M. H., & Cleland, W. W. (1982) Biochemistry 21, 5106-5114] has been used to study the mechanism of the oxidative decarboxylation catalyzed by 6-phosphogluconate dehydrogenase from yeast. 13C kinetic isotope effects of 1.0096 and 1.0081 with unlabeled or 3-deuterated 6-phosphogluconate, plus a 13C equilibrium isotope effect of 0.996 and a deuterium isotope effect on V/K of 1.54, show that the chemical reaction after the substrates have bound is stepwise, with hydride transfer preceding decarboxylation. The kinetic mechanism of substrate addition is random at pH 8, since the deuterium isotope effect is the same when either NADP or 6-phosphogluconate or 6-phosphogluconate-3-d is varied at fixed saturating levels of the other substrate. Deuterium isotope effects on V and V/K decrease toward unity at high pH at the same time that V and V/K are decreasing, suggesting that proton removal from the 3-hydroxyl may precede dehydrogenation. Comparison of the tritium effect of 2.05 with the other measured isotope effects gives limits of 3-4 on the intrinsic deuterium and of 1.01-1.05 for the intrinsic 13C isotope effect for C-C bond breakage in the forward direction and suggests that reverse hydride transfer is 1-4 times faster than decarboxylation.     

Kinetic and mechanistic studies of proline-mediated direct intermolecular aldol reactions

Reaction progress kinetic analysis of the proline-mediated intermolecular aldol reaction shows that the rate depends on the concentrations of both the donor ketone1 and the electrophilic aldehyde 2, implying that enamine formation cannot be rate-determining. The observed kinetics and deuterium isotope effects are consistent with formation of the product iminium species as the rate-determining step.     

Kinetic deuterium isotope effects on the N-demethylation of tertiary amides by cytochrome P-450

Liver microsomal cytochrome P-450 readily N-dealkylates N,N-dimethylamides. N-Methyl-N-hydroxymethyl amides were isolated as intermediates and characterized by gas chromatography-mass spectrometry as their trimethylsilyl ethers. Intramolecular kinetic deuterium isotope effects measured for the enzymic N-demethylation of a series of 12 aromatic and aliphatic N-methyl-N-trideuteriomethyl amides, RCON(CH3)CD3, varied from 3.6 to 6.9 but were independent of both amide bond rotation rate and substrate oxidation potential. These values, which represent a lower limit to the intrinsic isotope effect (Dkintrinsic), are significantly larger than those observed for anodic N-demethylation and are consistent with a mechanism involving hydrogen atom abstraction. On the other hand, with N,N-dimethylbenzamide the intermolecular kinetic deuterium isotope effects on Vmax and Vmax/Km were found to be much smaller (1.23 and 1.75, respectively) indicating substantial suppression of the intrinsic isotope effect. Such suppression indicates the occurrence of a rate-limiting step other than the isotopically sensitive step together with a strong commitment to catalysis.     

Design,synthesis, and biological evaluation of a novel series of peripheral-selective noradrenaline reuptake inhibitors—Part 2

Peripherally selective inhibition of noradrenaline reuptake is a novel mechanism for the treatment of stress urinary incontinence to overcome adverse effects associated with central action. Herein, we describe our medicinal chemistry approach to discover peripheral-selective noradrenaline reuptake inhibitors to avert the risk of P-gp-mediated DDI at the blood–brain barrier. We observed that steric shielding of the hydrogen-bond acceptors and donors (HBA and HBD) of compound 1 reduced the multidrug resistance protein 1 (MDR1) efflux ratio; however, the resulting compound 6, a methoxyacetamide derivative, was mainly metabolized by CYP2D6 and CYP2C19 in the in vitro phenotyping study, implying the risk of PK variability based on the genetic polymorphism of the CYPs. Replacement of the hydrogen atom with a deuterium atom in a strategic, metabolically hot spot led to compound 13, which was mainly metabolized by CYP3A4. To our knowledge, this study represents the first report of the effect of deuterium replacement for a major metabolic enzyme. The compound 13, N-{[(6S,7R)-7-(4-chloro-3-fluorophenyl)-1,4-oxazepan-6-yl]methyl}-2-[(2H³)methyloxy]acetamide hydrochloride, which exhibited peripheral NET selective inhibition at tested doses in rats, increased urethral resistance in a dose-dependent manner.     

Magnitude of intrinsic isotope effects in the dopamine beta-monooxygenase reaction

Intrinsic primary hydrogen isotope effects (kH/kD) have been obtained for the carbon-hydrogen bond cleavage step catalyzed by dopamine beta-monooxygenase. Irreversibility of this step is inferred from the failure to observe back-exchange of tritium from TOH into substrate under conditions of dopamine turnover; this result cannot be due to solvent inaccessibility at the enzyme active site, since we will demonstrate [Ahn, N., & Klinman, J. P. (1983) Biochemistry (following paper in this issue)] that a solvent-derived proton or triton must be at the enzyme active site prior to substrate activation. As shown by Northrop [Northrop, D. B. (1975) Biochemistry 14, 2644], for enzymatic reactions in which the carbon-hydrogen bond cleavage step is irreversible, comparison of D(V/K) to T(V/K) allows an explicit solution for kH/kD. Employing a double-label tracer method, we have been able to measure deuterium isotope effects on Vmax/Km with high precision, D(V/K) = 2.756 +/- 0.054 at pH 6.0. The magnitude of the tritium isotope effect under comparable experimental conditions is T(V/K) = 6.079 +/- 0.220, yielding kH/kD = 9.4 +/- 1.3. This result was obtained in the presence of saturating concentrations of the anion activator fumarate. Elimination of fumarate from the reaction mixture leads to high observed values for isotope effects on Vmax/Km, together with an essentially invariant value for kH/kD = 10.9 +/- 1.9. Thus, the large disparity between isotope effects, plus or minus fumarate, cannot be accounted for by a change in kH/kD, and we conclude a role for fumarate in the modulation of the partitioning of enzyme-substrate complex between catalysis and substrate dissociation. On the basis of literature correlations of primary hydrogen isotope effects and the thermodynamic properties of hydrogen transfer reactions, the very large magnitude of kH/kD = 9.4-10.9 for dopamine beta-monooxygenase suggests an equilibrium constant not very far from unity for the carbon-hydrogen bond cleavage step. This feature, together with the failure to observe re-formation of dopamine from enzyme-bound intermediate or product and overall rate limitation of enzyme turnover by product release, leads us to propose a stepwise mechanism for norepinephrine formation from dopamine in which carbon-hydrogen bond cleavage is uncoupled from the oxygen insertion step.     

Cytochrome P-450-catalyzed hydroxylation and carboxylic acid ester cleavage of Hantzsch pyridine esters

Cytochrome P-450 (P-450)-catalyzed oxidation of 2,6-dimethyl-4-phenyl-3,5-pyridinedicarboxylic acid diethyl ester gives rise to 2,6-dimethyl-4-phenyl-3,5-pyridinedicarboxylic acid monoethyl ester and to 2-hydroxymethyl-6-methyl-4-phenyl-3,5-pyridinedicarboxylic acid diethyl ester, identified in this work. A pyridine hydroxymethyl diester of the sort of the latter compound is novel; under acidic or dehydrating conditions the diester is readily converted to a cyclic lactone (2-hydroxymethyl-6-methyl-4-phenyl-3,5-pyridinedicarboxylic acid 5-ethyl ester lactone). 2,6-Dimethyl-4-phenyl-3,5-pyridinedicarboxylic acid monoethyl ester was not hydroxylated to form this hydroxymethyl compound or lactone, but 1,4-dihydro-2-hydroxymethyl-4-phenyl-6-methyl-3,5-pyridinedicarboxyli c acid diethyl ester was enzymatically oxidized to give both products. The rates of oxidative carboxylic ester cleavage and methyl hydroxylation varied among individual forms of P-450 tested. Experiments with 2H and 3H labels were used to estimate an intrinsic kinetic deuterium isotope effect of 15 for ethyl ester cleavage by rat liver P-450PB-B in a reconstituted system. Rat liver microsomal systems showed kinetic deuterium and tritium isotope effects of 8 and 11, respectively, and this deuterium isotope effect was not attenuated in either intra- or intermolecular competitive experiments. When deuterium was present in the ethyl (ester) groups, increases in the rate of 2-methyl hydroxylation were observed in rat liver microsomes and with purified P-450 beta NF-B (but not with P-450PB-B). Deuteration of the methyl groups gave rise to kinetic isotope effects of 7-11, but no increases were seen in the rates of ester cleavage. These studies and those on rates of substrate disappearance indicate that isotopically sensitive branching (metabolic switching) observed in these systems is not necessarily bidirectional.     

6-Phosphogluconate Dehydrogenase Mechanism: EVIDENCE FOR ALLOSTERIC MODULATION BY SUBSTRATE

The reductive carboxylation of ribulose-5-phosphate (Ru5P) by 6-phosphogluconate dehydrogenase (6PGDH) from Candida utilis was investigated using kinetic isotope effects. The intrinsic isotope effect for proton abstraction from Ru5P was found at 4.9 from deuterium isotope effects on V and V/K and from tritium isotope effects on V/K. The presence of 6-phosphogluconate (6PG) in the assay mixture changes the magnitude of the observed isotope effects. In the absence of 6PG ^D(V/K) and ^D(V) are 1.68 and 2.46, respectively, whereas the presence of 6PG increases ^D(V/K) to 2.84 and decreases ^D(V) to 1.38. A similar increase of ^T(V/K) is observed as 6PG builds up in the reaction mixture. These data indicate that in the absence of 6PG, a slow step, which precedes the chemical process, is rate-limiting for the reaction, whereas in the presence of 6PG, the rate-limiting step follows the isotope-sensitive step. Kinetic analysis of reductive carboxylation shows that 6PG at low concentrations decreases the K_m of Ru5P, whereas at higher concentrations, the usual competitive pattern is observed. These data indicate that full activity of 6PGDH is achieved when one subunit carries out the catalysis and the other subunit carries an unreacted 6PG. Thus, 6PG is like an allosteric activator of 6PGDH.     

Solid phase isotope exchange of deuterium and tritium for hydrogen in human recombinant insulin

Reaction of a high-temperature solid-phase catalytic isotope exchange in peptides and proteins under the action of the catalytically activated spillover hydrogen was studied. The reaction of human recombinant insulin with deuterium and tritium at 120–140°C resulted in an incorporation of 2–6 isotope hydrogen atoms per one insulin molecule. The distribution of the isotopic label by amino acid residues of the tritium-labeled insulin was determined by the oxidation of the protein S-S-bonds by performic acid, separation of polypeptide chains, their subsequent acidic hydrolysis, amino acid analysis, and liquid scintillation counts of tritium in the amino acids. The isotopic label was shown to be incorporated in all the amino acid residues of the protein, but the higher inclusion was observed for the FVNQHLCGSHLVE peptide fragment (B_1–13) of the insulin B-chain, and the His5 and His10 residues of this fragment contained approximately 45% of the whole isotopic label of the protein. Reduction of the S-S-bonds by 2-mercaptoethanol, enzymatic hydrolysis by glutamyl endopeptidase from Bacillus intermedius, and HPLC fractionation of the obtained peptides were also used for the analysis of the distribution of the isotopic label in the peptide fragments of the labeled insulin. Peptide fragments which were formed after the hydrolysis of the Glu-Xaa bond of the B-chain were identified by mass spectrometry. The mass spectrometric analysis of the isotopomeric composition of the deuterium-labeled insulin demonstrated that all the protein molecules participated equally in the reaction of the solid-phase hydrogen isotope exchange. The tritium-labeled insulin preserved the complete physiological activity.     

Tritium incorporation and retention in photosynthesizing algae

The ratios of incorporation and retention of tritium compared to protium into metabolites in Chlorella pyrenoidosa and in Anacystis nidulans growing in water labeled with tritium have been determined. The algae were continuously supplied during growth with CO₂ labeled with ¹⁴CO₂, and the ¹⁴C content of metabolites were used to determined their concentrations. The tritium/protium ratios (R) of metabolites in Chlorella were determined following growth at 10 °C, 20 °C and 25 °C.As previously reported, variations in R in Chlorella, range from 0.5–0.7 for most metabolites, to values of R around 1 for metabolites of the tricarboxylic acid pathway. The R value for fumarate has now been measured. The increased R values for tricarboxylic acid cycle intermediates and related amino acids can be accounted for in terms of specific isotope effects of several enzyme-mediated steps. Very different R values for certain metabolites were found in A. nidulans. For example, R for citrate was 1.81 (the highest value observed in these studies) while aspartate was only 0.59, comparable to other metabolites in both organisms not related to the tricarboxyclic acid cycle. This lower value for aspartate is explainable in terms of the in complete tricarboxylic acid cycle in A. nidulans.No significant differences in R values for C. pyrenoidosa grown at 20 °C and 25 °C were observed, but in cells grown at 10 °C, there was a small but significant increase in R for tricarboxylic acid cycle metabolites.If the increase in R from sugar phosphates to tricarboxylic acid cycle intermediates seen in these two types of algae may be taken as an indication of likely discriminatory retention of tritium in organisms higher in the food chain, it would appear that no serious concentration of tritium due to isotopic discrimination should occur in the biosphere. However, research workers using compounds labeled with hydrogen isotopes for studies of in vivo metabolism should take into account the likelihood of such discriminatory uptake and retention during specific metabolic steps.     

Isotope ratios of cellulose from plants having different photosynthetic pathways

Hydrogen and carbon isotope ratios of cellulose nitrate and oxygen isotope ratios of cellulose from C₃, C₄, and Crassulacean acid metabolism (CAM) plants were determined for plants growing within a small area in Val Verde County, Texas. Plants having CAM had distinctly higher deuterium/hydrogen (D/H) ratios than plants having C₃ and C₄ metabolism. When hydrogen isotope ratios are plotted against carbon isotope ratios, each photosynthetic mode separates into a distinct cluster of points. C₄ plants had many D/H ratios similar to those of C₃ plants, so that hydrogen isotope ratios cannot be used to distinguish between these two photosynthetic modes. Portulaca mundula, which may have a modified photosynthetic mode between C₄ and CAM, had a hydrogen isotope ratio between those of the C₄ and CAM plants. When oxygen isotope ratios are plotted against carbon isotope ratios, no distinct clustering of the C₄ and CAM plants occurs. Thus, oxygen isotope ratios are not useful in distinguishing between these metabolic modes. A plot of hydrogen isotope ratios versus oxygen isotope ratios for this sample set shows considerable overlap between oxygen isotope ratios of the different photosynthetic modes without a concomitant overlap in the hydrogen isotope ratios of CAM and the other two photosynthetic modes. This observation is consistent with the hypothesis that higher D/H ratios in CAM plants relative to C₃ and C₄ plants are due to isotopic fractionations occurring during biochemical reactions.     

Deuterium isotope shifts for backbone 1H, 15N and 13C nuclei in intrinsically disordered protein α-synuclein

Intrinsically disordered proteins (IDPs) are abundant in nature and characterization of their potential structural propensities remains a widely pursued but challenging task. Analysis of NMR secondary chemical shifts plays an important role in such studies, but the output of such analyses depends on the accuracy of reference random coil chemical shifts. Although uniform perdeuteration of IDPs can dramatically increase spectral resolution, a feature particularly important for the poorly dispersed IDP spectra, the impact of deuterium isotope shifts on random coil values has not yet been fully characterized. Very precise ²H isotope shift measurements for ¹³C^??, ¹³C^??, ¹³C??, ¹⁵N, and ¹H^N have been obtained by using a mixed sample of protonated and uniformly perdeuterated ??-synuclein, a protein with chemical shifts exceptionally close to random coil values. Decomposition of these isotope shifts into one-bond, two-bond and three-bond effects as well as intra- and sequential residue contributions shows that such an analysis, which ignores conformational dependence, is meaningful but does not fully describe the total isotope shift to within the precision of the measurements. Random coil ²H isotope shifts provide an important starting point for analysis of such shifts in structural terms in folded proteins, where they are known to depend strongly on local geometry.