Tritium Isotope Effects Research Articles

34] Deduction of kinetic mechanisms from primary Hydrogen isotope effects: Dopamine β-monooxygenase —A case history

Publisher Summary An in-depth understanding of the chemical mechanism would include a description of chemical intermediates as well as the structure of the metastable enzyme transition-state complex(es). Kinetic isotope effects provide a uniquely versatile, nonperturbing probe of both kinetic and chemical mechanisms in enzyme-catalyzed reactions. Employing dopamine β-monooxygenase as a model system, this chapter illustrates (1) the determination of the order of substrate addition in multisubstrate enzyme reactions from the variation of isotope effects on Vm/Km for labeled substrate as a function of the concentration of second, unlabeled substrate. (2) The successful measurement of both deuterium and tritium isotope effects on Vm/Km with the requisite accuracy to allow the calculation of an intrinsic isotope effect with an uncertainty of

Secondary tritium isotope effects, a useful mechanistic probe for the study of acetoxyl participation and transfer

Tritium labelling on the acetoxyl methyl group has been used to probe the mode of incorporation of acetate into cinnamyl acetate in the solvolyes of cinnamyl chloride and cinnamylmercuric acetate, and in the allylic oxidation of allylbenzene with mercuric acetate.

Primary deuterium and tritium isotope effects upon V/K in the liver alcohol dehydrogenase reaction with ethanol.

The primary isotope effect upon V/K when ethanol stereospecifically labeled with deuterium or tritium is oxidized by liver alcohol dehydrogenase has been measured between pH 6 and 9. The deuterium isotope effect was obtained with high reproducibility by the use of two different radioactive tracers, viz. 14C and 3H, to follow the rate of acetaldehyde formation from deuterium-labeled ethanol and normal ethanol, respectively. Synthesis of the necessary labeled compounds is described in this and earlier work referred to. V/K isotope effects for both tritium and deuterium have been measured with three different coenzymes, NAD+, thio-NAD+, and acetyl-NAD+. With NAD+ at pH 7, D(V/K) was 3.0 and T(V/K) was 6.5. With increasing pH, these values decreased to 1.5 and 2.5 at pH 9. The intrinsic isotope effect evaluated by the method of Northrop [Northrop, D.B. (1977) in Isotope Effects on Enzyme-Catalyzed Reactions (Cleland, W. W., O'Leary, M, H., & Northrop, D. B., Eds.) pp 112-152, University Park Press, Baltimore] varies little with pH. It amounts to about 10 with NAD+ and about 5 with the coenzyme analogues. Commitment functions and their dependence upon pH calculated in this connection appear to be in agreement with known kinetic parameters of liver alcohol dehydrogenase. This assay method was also applied in vivo in the rat. Being a noninvasive method because only trace amounts of isotopes are needed, it may yield information about alternative routes of ethanol oxidation in vivo. In naive rats at low concentrations of ethanol, it confirms the discrete role of the non alcohol dehydrogenase systems.

Stereochemistry and kinetic isotope effects in the decarboxylation of S-adenosylmethionine catalyzed by the pyruvyl enzyme, S-adenosylmethionine decarboxylase.

S-Adenosyl-5'-3-methylthio[1-3H]propylamine was prepared by decarboxylation of S-adenosylmethionine in tritiated water, catalyzed by S-adenosylmethionine decarboxylase from Escherichia coli. Degradation of this product to 3-methylthio[1-3H]propylamine followed by oxidation in a coupled assay system involving pea seedling amine oxidase, catalase, and aldehyde dehydrogenase indicated less than 4% of total counts in water and greater than 75% in NADH plus NAD+. These results allow the assignment of tritium in 3-methylthiopropylamine to the 1-R configuration, establishing that S-adenosylmethionine decarboxylase, a pyruvate-containing decarboxylase, operates via a retentive mode. Tabulation of the available stereochemical results for two pyruvate-containing and three pyridoxal phosphate-dependent amino acid decarboxylases indicates that stereochemistry has been conserved both within each class of amino acid decarboxylases and between structurally distinct classes of decarboxylases. Comparison of the specific activity of 3-methylthio[1-3H]propylamine to the tritiated water employed in the decarboxylation reaction leads to a primary tritium isotope effect of 4.5 under conditions of substrate concentration far in excess of Km. Although a portion of the tritium in 3-methylthiopropylamine could have arisen through an enzyme-catalyzed exchange into decarboxylated adenosylmethionine (leading to an underestimate of the kinetic tritium isotope effect), we are unable to detect enzymatic loss of tritium from decarboxylated S-adenosylmethionine. The magnitude of the observed isotope effect is discussed in the context of a kinetically significant exchange of an active site residue with solvent in the present of enzyme-bound S-adenosylmethionine.

Kinetic hydrogen isotope effects in intermolecular hydride transfer from arylalkanes to 9-arylfluoren-9-yl cations

Rates of hydride-ion transfer to a series of 9-phenylfluoren-9-yl cations, substituted in the phenyl group, from triphenylmethane and 4,4′-dimethoxydiphenylmethane have been measured at 30°C in trifluoroacetic acid solution containing 6% v/v acetic acid. Deuterium kinetic isotope effects have been measured directly and show evidence of a broad maximum, the peak value occurring when the difference in pKR+ values between the product and reactant carbenium ions is around zero. Application of Marcus theory suggests that the intrinsic barrier to hydride transfer in these systems is substantially larger than for hydride transfer from formate ion to a series of triphenylmethyl cations. A limited series of measurements has also been made of tritium kinetic isotope effects on hydride transfer from 4,4′-dimethoxydiphenylmethane to 9-arylfluoren-9-yl cations and these have been used to derive deuterium–tritium isotope effects. The relevance of the results to the question of the origin of isotope-effect maxima is briefly discussed.

Tritium/protium discrimination in reduction of cyclopropenium ion by sodium borohydride does not identify the rate-determining step

Intramolecular isotope discrimination vitiates an attempt to deduce from the observation of a tritium isotope effect in borohydride reduction of a cyclopropenium ion that hydride transfer is at least partially rate determining.

The tritium kinetic isotope effect for the iodination of L-(5-3H)-3-Iodotyrosine

The kinetic tritium isotope effect (kH/kT) for the iodination of L-(5-3H)-3-iodotyrosine at pH 7·5 was found to be 4·29�0.25 with molecular iodine and 4·50�0.26 with chloramine-T/potassium iodide. This effect establishes that the rate-limiting step is proton removal by the general base. A novel method for measuring the fraction of tritiated water by high performance liquid chromatography is described.

ChemInform Abstract: MECHANISMS OF ELIMINATION REACTIONS. 32. TRITIUM ISOTOPE EFFECTS AND TUNNEL EFFECTS IN THE REACTION OF 2,2‐DIPHENYLETHYL‐2‐T DERIVATIVES WITH VARIOUS BASES

Chemischer InformationsdienstVolume 11, Issue 52 Preparative Organic Chemistry ChemInform Abstract: MECHANISMS OF ELIMINATION REACTIONS. 32. TRITIUM ISOTOPE EFFECTS AND TUNNEL EFFECTS IN THE REACTION OF 2,2-DIPHENYLETHYL-2-T DERIVATIVES WITH VARIOUS BASES S. B. KALDOR, S. B. KALDORSearch for more papers by this authorM. E. FREDENBURG, M. E. FREDENBURGSearch for more papers by this authorW. H. JUN. SAUNDERS, W. H. JUN. SAUNDERSSearch for more papers by this author S. B. KALDOR, S. B. KALDORSearch for more papers by this authorM. E. FREDENBURG, M. E. FREDENBURGSearch for more papers by this authorW. H. JUN. SAUNDERS, W. H. JUN. SAUNDERSSearch for more papers by this author First published: December 30, 1980 https://doi.org/10.1002/chin.198052118Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume11, Issue52December 30, 1980 RelatedInformation

Deduction of kinetic mechanism in multisubstrate enzyme reactions from tritium isotope effects. Application to dopamine beta-hydroxylase.

Primary tritium isotope effects have been measured for the hydroxylation of [2-3H]dopamine catalyzed by dopamine beta-hydroxylase. Experimental values vary from 8.8 +/- 1.4 at 0.02 mM oxygen to 4.1 +/- 0.6 at 1.0 mM oxygen. It is shown that the observed dependence of the isotope effect on oxygen concentration provides unequivocal evidence for a kinetically significant dissociation of both dopamine and oxygen from enzyme . ternary complex. This approach, which is applicable to any multisubstrate enzyme characterized by detectable kinetic isotope effects, provides an alternate to classical methods for the elucidation of kinetic order in enzyme-catalyzed reactions.

Mechanisms of elimination reactions. 32. Tritium isotope effects and tunnel effects in the reaction of 2,2-diphenylethyl-2-t derivatives with various bases

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTMechanisms of elimination reactions. 32. Tritium isotope effects and tunnel effects in the reaction of 2,2-diphenylethyl-2-t derivatives with various basesSheena B. Kaldor, Mark E. Fredenburg, and William H. Saunders Jr.Cite this: J. Am. Chem. Soc. 1980, 102, 20, 6296–6299Publication Date (Print):September 1, 1980Publication History Published online1 May 2002Published inissue 1 September 1980https://doi.org/10.1021/ja00540a020RIGHTS & PERMISSIONSArticle Views121Altmetric-Citations3LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (576 KB) Get e-Alerts Get e-Alerts

Primary tritium isotope effect on hydride transfer from sodium borohydride to cyclopropenium ion

Reduction of a cyclopropenium perchlorate with tritium-labeled sodium borohydride shows a primary kinetic isotope effect, suggesting that hydride transfer is at least partially rate-determining and thus demonstrating that formation of the encounter complex between cation and hydride is reversible.

Thymidylate synthetase. Catalysis of dehalogenation of 5-bromo- and 5-iodo-2'-deoxyuridylate.

Tymidylate synthetase catalyzes the facile dehalogenation of 5-bromo-2'-deoxyuridylate (BrdUMP) and 5-iodo-2'-deoxyuridylate )IdUMP) to give 2'-deoxyuridylate (dUMP), the natural substrate of the enzyme. The reaction does not require folate cofactors and stoichiometrically consumes 2 equiv of thiol. In addition to dUMP, a minor product is formed during the debromination of BrdUMP which has been identified as a 5-alkylthio derivative formed by displacement of bromide ion by thiolate. The reaction has been found to proceed with a substantial alpha-secondary inverse tritium isotope effect (kT/kH = 1.212--1.258) with [2-14C,6-3H]-BrdUMP as the substrate. Similarly, an inverse tritiumisotope effect of 1.18 was observed in the nonenzymatic chemical counterpart of this reaction, the cysteine-promoted dehalogenation of [2-14C,6-3H]-5-bromo-2'-deoxyuridine. Previous evidence for the mechanism of action of this enzyme has rested largely on chemical model studies and on information obtained from its stoichiometric interaction with the inhibitor 5-fluoro-2'-deoxyuridylate. The magnitude of the secondary isotope effect during the enzymatic dehalogenation described here provides direct proof for nucleophilic catalysis and formation of 5,6-dihydroprimidine intermediates in a reaction catalyzed by thymidylate synthetase.

Stereochemistry and kinetic isotope effects in the bovine plasma amine oxidase catalyzed oxidation of dopamine

The stereochemistry of the bovine plasma amine oxidase catalyzed oxidation of 2-(3,4-dihydroxyphenyl)-ethylamine (domapine) has been investigated by comparing 3H/14C ratios of 3,4-dibenzyloxyphenethyl alcohols, derived from 3,4-dihydroxyphenylacetaldehydes, to starting dopamines chirally labeled at C-1 and C-2. The oxidation of [2RS-3H]-, [2R-3H]-, and [2S-3H]dopamine leads to products which have retained 53, 59, and 47% of their tritium. Similarly, oxidation of [1RS-3H]-, [1R-3H]-, and [1S-3H]dopamine leads to an 80, 80, and 92% retention of tritium. The configurational purity of tritium at C-2 of dopamine and C-1 of the dopamine precursor 3-methoxy-4-hydroxyphenethylamine has been confirmed employing dopamine-beta-hydroxylase (specific for the pro-R hydrogen at C-2) and pea seedling amine oxidase (specific for the pro-S hydrogen at C-1). In addition, chromatographically resolved isozymes of bovine plasma amine oxidase have been demonstrated to lead to the same stereochemical result as pooled enzyme fractions. We have been able to rule out carbon interchange and tritium transfer in the ethylamine side chain of dopamine as the source of the apparent nonstereospecificity. Estimated primary tritium isotope effects are 1 for [2-3H]dopamines and 5--6 and 26--34 for [1R-3H]- and [1S-3H]dopamine, respectively. We propose the presence of alternate dopamine binding modes, characterized by absolute but opposing stereochemistries and differential primary tritium isotope effects at C-1.

A deuterium-labeling method for the assignment of histidine nuclear magnetic resonance peaks of proteins

A tritium labelling method involving differential tritium exchange at the C-2 H position of histidines and /sup 1/H NMR of differentially deuterated proteins can be a general method for the assignment of the histidine NMR peaks. In the present report this method is modified by replacing the tritium with deuterium, which eliminates ambiguities arising from the tritium isotope effect. In the deuterium labelling method, differentially deuterated proteins are cleaved by trypsin into smaller peptides each containing a single histidine residue, which are separated by chromatography. The method was applied to the Bence Hones dimer Ak which contains two histidine residues in the constant domain of each of the light chains. The decay of the NMR peaks with time allowed the assignment of one peak to His/sup 189/ and the other to His/sup 198/. (JSR)

The Biosynthesis of Long-Chain Fatty Acids. Stereochemical Differentiation in the Enzymic Incorporation of Chiral Acetates

1 Acetyl-CoA carboxylase and fatty acid synthetase were purified from chicken liver. Fatty acid synthetase was also purified from bakers' yeast. 2 The corresponding CoA thiol esters were prepared enzymically from R-[2H1, 3H1]acetate, S-[2H1, 3H1]acetate and [3H1]acetate, using a linked acetate kinase/phosphotransacetylase system. [2-14C]Acetate was added to the tritiated specimens to provide doubly-labelled products, which were purified by column chromatography on DEAE-cellulose. 3 The three acetyl-CoA specimens thus obtained were incubated separately in a combined acetyl-CoA carboxylase/fatty acid synthetase system containing the cofactors necessary for synthesis of long-chain fatty acids. 4 The fatty acids formed were extracted into n-pentane, methylated, separated by gas-liquid chromatography and the 3H/14C isotope ratio determined. 5 In this manner it was shown, using the chicken liver synthetase, that in palmitic acid a higher proportion of tritium was retained from S-[2-14C, 2H1, 3H1]acetyl-CoA than from the R-(2-14C, 2H1, 3H1)-labelled thiol ester, the non-chiral (2-14C, 3H1)-labelled substrate giving an intermediate result. 6 This result was confirmed using the fatty acid synthetase preparation purified from bakers' yeast in place of the corresponding chicken liver enzyme. 7 In a separate experiment it was shown that the intramolecular tritium isotope effect in the carboxylation of [3H1]acetyl-CoA is normal, but small. The slightly smaller, normal deuterium isotope effect calculated from this was shown to be in good agreement with the value expected, based on the experiments on tritium incorporation from chiral acetyl-CoA. 8 It is demonstrated that partial exchange of carbon-bound hydrogen must occur on the synthetase during the enzymic synthesis of fatty acids from malonyl thiol esters and that the extent of this exchange differs in synthetases of different origin. 9 A theoretical treatment of the effect of partial hydrogen exchange on the chirality of asymmetrically labelled methylene groups is given. 10 The results indicate the existence of an overall stereospecificity in the reactions involved in the de novo biosynthesis of long-chain fatty acids in different cell types.

Isotope effects in hydrogen atom transfers. 9. Neighboring group participation

The tritium isotope effects in hydrogen abstraction by bromine atoms from HC(Me)/sub 2/CH/sub 2/X have been measured in the photobromination reaction by a technique which measures the specific activity of the HBr product. At 1/sup 0/C and with the reverse reaction suppressed or irrelevant the values for k/sub H//k/sub T/ are 3.9, 8.2, and 11.6 for X = CH/sub 3/, Cl, and Br, respectively. An isotope effect of 3.5 for X = H was also measured, but the contribution of the reverse reaction was not established. The relatively large isotope effect for X = Br is interpreted in terms of a weakening of the CH bond by neighboring bromine participation. The smaller effect of chlorine may correspond to a minor participation, but the unfavorable inductive effect of the chlorine is also a factor. The case of X = Br is the microscopic reverse of the addition of HBr to 2-methylpropene, and the results are compared with isotope effects in the addition to HBr to olefins. The measurement of isotope effect is, within some described limitations, a convenient and general method for detecting neighboring group participation in hydrogen atom abstractions.

Isotope effects in hydrogen atom transfers. V. The reactions of olefins with mercaptans

Tritium isotope effects have been measured for the hydrogen atom transfer R/sub 1/R/sub 2/CCH/sub 2/SR/sub 3/ + HSR/sub 3/ ..-->.. R/sub 1/R/sub 2/CHCH/sub 2/SR/sub 3/ + SR/sub 3/ by comparing the tritium content of excess mercaptan with that of its addition product to the olefin R/sub 1/R/sub 2/C = CH/sub 2/. Several somewhat obscure sources of error in earlier work are uncovered; elimination of these errors results in only minor revision of the values except for the addition of 1,1-diarylethylenes to thiophenol, where two new values of k/sub H//k/sub T/ are much larger than at first thought. The isotope effect in the reaction of mesitylenethiol with trityl radical is also reported; the value of k/sub H//k/sub T/ at 25/sup 0/C is 25, almost twice the value in the thiophenol-trityl radical reaction, and a steric enhancement of tunnelling is indicated. (auth)

Human placental 3beta-hydroxysteroid dehydrogenase: delta5-isomerase. Demonstration of an intermediate in the conversion of 3beta-hydroxypregn-5-en-20-one to pregn-4-ene-3,20-dione.

3beta-Hydroxypregn-5-en-20-one (pregnenolone) and NAD+ were incubated with a solubilized preparation of the coupled enzyme 3beta-hydroxysteroid:NAD(P) oxidoreductase-3-ketosteroid delta4,delta5-isomerase (3beta-hydroxysteroid dehydrogenase: delta5-isomerase) from the mitochondrial fraction of human placenta. Unconverted pregnenolone, pregn-4-ene-3,20-dione (rogesterone), and a small but detectable amount of pregn-5-ene-3,20-dione were isolated from the medium by Sephadex LH-20 chromomatography. The identification of pregn-5-ene-3,20-dione, confirmed by mass fragmentography, has provided the first direct evidence for the formation of the hypothetical delta5,3-ketone intermediate in the conversion of pregnenolone to progesterone. When tritium-labeled pregnenolone and [4-14C]pregnenolone were incubated simultaneously the 3H:14C ratio in isolated pregn-5-ene-3,20-dione was 4.6 times greater than in isolated progesterone and pregnenolone, indicating a kinetic isotope effect in the enzymatic isomerization of tritium-labeled pregn-5-ene-3,20-dione. Exposure of the enzyme to two steroids which inhibit the overall enzyme reaction, 2alpha-cyano-17beta-hydroxy-4,4,17alpha-trimethylandrost-5-en-3-one (cyanoketone) and 3-hydroxyestra-1,3,5(10),6,8-pentaen-17-one (equilenin), increased the relative yield of labeled pregn-5-ene-3,20-dione as well as the recovery of radioactivity remaining as unconverted pregnenolone, suggesting that both the dehydrogenase and isomerase activities were inhibited. Exposure of the enzyme to equilenin increased the ratio of isolated pregn-5-ene-3,20-dione radioactivity to progesterone radioactivity as progesterone synthesis was inhibited. Equilenin also diminished the tritium isotope effect on the isomerase reaction. Both findings suggest that it is possible to inhibit the isomerase to a greater extent than the dehydrogenase. In order to measure the rate of progesterone produced by the coupled enzymes, we have modified a radiochemical method which involves precipitation of pregnenolone by digitonin. Digitonin precipitation proved to be effective in separating unconverted pregnenolone from the steroid products of both enzyme reactions, progesterone and pregn-5-ene-3,20-dione. Neither the steroidal inhibitors nor the kinetic isotope effect altered the accuracy of the method for routine measurement of the overall rate of conversion of delta5,3beta-hydroxysteroid to delta4,3-ketosteroid.

α-Secondary tritium isotope effects in the aqueous hydrolysis of glycopyranosides of N-acetyl-β- d-glucosamine

Secondary tritium isotope effects were used to study the aqueous hydrolysis of a series of α- and β-glycopyranosides of N-acetyl- d-glucosamine. The magnitude of the secondary tritium isotope effects, and their dependence on the structure of the aglycone, are compatible with the carbonium ion mechanism suggested for the specific acid catalysis of these compounds by Piszkiewicz and Bruice (1–3). The secondary tritium isotope effect determined for the spontaneous hydrolysis of the p-nitrophenyl-2-acetamido-2-deoxy-β- d-glucopyranoside is not consistent with an intramolecular, nucleophilic displacement mechanism. A mechanism involving the equilibrium formation of a carbonium ion-anion pair is proposed. The relevance of these model studies to hydrolysis of oligosaccharides of N-acetyl- d-glucosamine by lysozyme is discussed.

The tritium isotope effect of sn-glycerol 3-phosphate oxidase and the effects of clofenapate and N-(2-benzoyloxyethyl)norfenfluramine on the esterification of glycerol phosphate and dihydroxyacetone phosphate by rat liver mitochondria.

1. Owing to a (3)H isotope effect, the mitochondrial sn-glycerol 3-phosphate oxidase (EC 1.1.99.5) had a mean activity which was 8.4 times less with sn-[2-(3)H]-rather than with sn-[1-(14)C]glycerol 3-phosphate as a substrate. 2. A method for measuring the simultaneous synthesis of lipid from glycerol phosphate and dihydroxyacetone phosphate in rat liver mitochondria is described. 3. The lipid synthesized by rat liver mitochondria from sn-[1-(14)C]glycerol 3-phosphate was mainly phosphatidate and lysophosphatidate, whereas that synthesized from dihydroxy[1-(14)C]acetone phosphate was mainly acyldihydroxyacetone phosphate. 4. Additions of NADPH facilitated the conversion of acyldihydroxyacetone phosphate into lysophosphatidate and phosphatidate. 5. Hydrazine (1.4mm) or KCN (1.4mm) inhibited the synthesis of lipids from dihydroxyacetone phosphate but not from glycerol phosphate. 6. Clofenapate (1-2.5mm) inhibited the synthesis of lipids from dihydroxyacetone phosphate but slightly stimulated synthesis from glycerol phosphate. 7. The methanesulphonate of N-(2-benzoyloxyethyl)norfenfluramine, at 0.25-0.75mm, inhibited lipid synthesis from both glycerol phosphate and dihydroxyacetone phosphate.