Deuterium isotope effects on noncovalent interactions between molecules

The topic of deuterium isotope effects is usually concerned with the effects on chemical reactions that are caused by the substitution of deuterium atoms for protium, or hydrogen, atoms in a molecule. These effects include changes in the rate of cleavage of covalent bonds to deuterium, or to an atom located adjacent to deuterium, in a reactant molecule. Deuterium isotope effects on other, noncovalent, interactions between molecules are known to occur, but they are generally considered to be insignificant, especially in biological experiments where deuterium substituted molecules are used as tracers. Noncovalent interactions between molecules include hydrogen bonding, and ionic and van der Waals interactions. This article reviews evidence for deuterium isotope effects on noncovalent interactions, with an emphasis on binding interactions between molecules of biological interest, but also including examples of nonbiological molecules in order to demonstrate the generality of these effects. The reality of this effect relies on the assumption that the only difference between the isotopomers considered is the presence of deuterium or hydrogen; there are no impurities present. The physical basis of the effect may be due to differences in the polarities and/or sizes of deuterated versus nondeuterated isomers, and the extent of a deuterium isotope effect on a noncovalent interaction depends on the site of deuteration within a biomolecule. The presence of this effect requires careful interpretation of results obtained in experiments with deuterium labeled compounds.     

Deuterium isotope effects on the rates of steroid--glucocorticoid receptor interactions

Rate constants of association and dissociation of several steroids to and from glucocorticoid receptor protein of chick thymus cytosol were determined in water and deuterium oxide. Substitution of deuterium for hydrogen did not influence association rate constants. Dissociation rate constants decreased about twofold in deuterium oxide in case of steroids containing an 11-beta-hydroxyl group but remained unchanged if the steroid had no hydroxyl or had an alpha-hydroxyl group at position 11. Presence of molybdate ions decreased but did not abolish the deuterium isotope effect. These findings suggest that the 11-beta-hydroxyl group, known to be present in every optimal glucocorticoid agonist molecule, participates in a kinetically relevant hydrogen bond, and that this hydrogen bond may have a role in glucocorticoid action.     

Deuterium isotope effects in the unusual addition of toluene to fumarate catalyzed by benzylsuccinate synthase

The first step in the anaerobic metabolism of toluene is a highly unusual reaction: the addition of toluene across the double bond of fumarate to produce (R)-benzylsuccinate, which is catalyzed by benzylsuccinate synthase. Benzylsuccinate synthase is a member of the glycyl radical-containing family of enzymes, and the reaction is initiated by abstraction of a hydrogen atom from the methyl group of toluene. To gain insight into the free energy profile of this reaction, we have measured the kinetic isotope effects on Vmax and Vmax/Km when deuterated toluene is the substrate. At 30 degrees C the isotope effects are 1.7 +/- 0.2 and 2.9 +/- 0.1 on Vmax and Vmax/Km, respectively; at 4 degrees C they increase slightly to 2.2 +/- 0.2 and 3.1 +/- 0.1, respectively. We compare these results with the theoretical isotope effects on Vmax and Vmax/Km that are predicted from the free energy profile for the uncatalyzed reaction, which has previously been computed using density functional theory [Himo, F. (2002) J. Phys. Chem. B 106, 7688-7692]. The comparison allows us to draw some conclusions on how the enzyme may catalyze this unusual reaction.     

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.     

Mechanism of inhibition of catalase by nitro and nitroso compounds

Dinitrosyl iron complexes (DNIC) with thiolate ligands and S-nitrosothiols, which are NO and NO+ donors, share the earlier demonstrated ability of nitrite for inhibition of catalase. The efficiency of inhibition sharply (by several orders in concentration of these agents) increases in the presence of chloride, bromide, and thiocyanate. The nitro compounds tested--nitroarginine, nitroglycerol, nitrophenol, and furazolidone--gained the same inhibition ability after incubation with ferrous ions and thiols. This is probably the result of their transformation into DNIC. None of these substances lost the inhibitory effect in the presence of the well known NO scavenger oxyhemoglobin. This fact suggests that NO+ ions rather than neutral NO molecules are responsible for the enzyme inactivation due to nitrosation of its structures. The enhancement of catalase inhibition in the presence of halide ions and thiocyanate might be caused by nitrosyl halide formation. The latter protected nitrosonium ions against hydrolysis, thereby ensuring their transfer to the targets in enzyme molecules. The addition of oxyhemoglobin plus iron chelator o-phenanthroline destroying DNIC sharply attenuated the inhibitory effect of DNIC on catalase. o-Phenanthroline added alone did not influence this effect. Oxyhemoglobin is suggested to scavenge nitrosonium ions released from decomposing DNIC, thereby preventing catalase nitrosation. The mixture of oxyhemoglobin and o-phenanthroline did not affect the inhibitory action of nitrite or S-nitrosothiols on catalase.     

Deuterium kinetic isotope effects in the p-side pathway for quinol oxidation by the cytochrome b(6)f complex.

Plastoquinol oxidation and proton transfer by the cytochrome b(6) f complex on the lumen side of the chloroplast thylakoid membrane are mediated by high and low potential electron transport chains. The rate constant for reduction, k(bred), of cytochrome b(6) in the low potential chain at ambient pH 7.5-8 was twice that, k(fred), of cytochrome f in the high potential chain, as previously reported. k(bred) and k(fred) have a similar pH dependence in the presence of nigericin/nonactin, decreasing by factors of 2.5 and 4, respectively, from pH 8 to an ambient pH = 6, close to the lumen pH under conditions of steady-state photosynthesis. A substantial kinetic isotope effect, k(H2O)/k(D2O), was found over the pH range 6-8 for the reduction of cytochromes b(6) and f, and for the electrochromic band shift associated with charge transfer across the b(6)f complex, showing that isotope exchange affects the pK values linked to rate-limiting steps of proton transfer. The kinetic isotope effect, k(bred)(H2O)/k(bred) (D2O) approximately 3, for reduction of cytochrome b in the low potential chain was approximately constant from pH 6-8. However, the isotope effect for reduction of cytochrome f in the high potential chain undergoes a pH-dependent transition below pH 6.5 and increased 2-fold in the physiological region of the lumen pH, pH 5.7-6.3, where k(fred)(H2O)/k(fred)(D2O) approximately 4. It is proposed that a rate-limiting step for proton transfer in the high potential chain resides in the conserved, buried, and extended water chain of cytochrome f, which provides the exit port for transfer of the second proton derived from p-side quinol oxidation and a "dielectric well" for charge balance.     

Deuterium isotope effects on toluene metabolism. Product release as a rate-limiting step in cytochrome P-450 catalysis

Liver microsomes from phenobarbital-induced rats oxidize toluene to a mixture of benzyl alcohol plus o-, m- and p-cresol (ca. 69:31). Stepwise deuteration of the methyl group causes stepwise decreases in the yield of benzyl alcohol relative to cresols (ca. 24:76 for toluene-d3). For benzyl alcohol formation from toluene-d3 DV = 1.92 and D(V/K) = 3.53. Surprisingly, however, stepwise deuteration induces stepwise increases in total oxidation, giving rise to an inverse isotope effect overall (DV = 0.67 for toluene-d3). Throughout the series (i.e. d0, d1, d2, d3) the ratios of cresol isomers remain constant. These results are interpreted in terms of product release for benzyl alcohol being slower than release of cresols (or their epoxide precursors), and slow enough to be partially rate-limiting in turnover. Thus metabolic switching to cresol formation causes a net acceleration of turnover.     

Nitrogen isotope effects in the assimilation of inorganic nitrogenous compounds by marine diatoms

Nitrogen isotope fractionation in the assimilation of inorganic nitrogenous compounds was studied using marine diatoms (Phaeodactylum tricornutum and Chaetoceros sp.). The isotopic composition (δ¹⁵N) of the diatoms ranged from 7 to ‐18‰ relative to that of the nitrogen source, i.e., ammonium, nitrite, or nitrate. When the growth was light‐limited, the isotope fractionation in nitrate assimilation was inversely correlated with the growth rate. The highest fractionation factor of 1.016 was obtained when the growth rate was as low as 0.025 day^‐1. Fractionation was negligible when the growth, rate was higher than 1 day^‐1. A steady‐state kinetic model was applied to explain the isotope fractionation in nitrate assimilation. The nitrogen isotope fractionation primarily takes place at the step of N‐O bond breaking in nitrate reduction to nitrite. The extent of the isotope fractionation associated with the nitrate uptake is very small, and barely exceeds the limit of detection.     

Deuterium isotope effect in bioactivation and hepatotoxicity of chloroform

Cytochrome P-450 appears to catalyze the $\text{in}$$\text{vitro}$ formation of phosgene (COCl₂) from chloroform (CHCl₃) in rat liver microsomes, since this reaction is NADPH dependent and inhibited by carbon monoxide and SKF 525-A. Moreover, the cleavage of the C-H bond appears to be the rate-determining step in this process since deuterium labeled chloroform (CDCl₃) is biotransformed into COCl₂ slower than is CHCl₃. CDCl₃ was also less hepatotoxic than CHCl₃ suggesting that a similar pathway of metabolism is responsible for the hepatotoxic properties of chloroform.     

Deuterium isotope effect in the transamination of p-tyrosine by rat liver tyrosine transaminase

The rate of transamination of p-tyrosine catalyzed by rat liver soluble tyrosine aminotransferase (E.C. 2.6.1.5.) was significantly reduced when the hydrogen at the alpha-carbon position is replaced by deuterium or when the reactions were conducted in 2H2O. The cleavage of carbon-hydrogen bond at alpha-carbon position is at least partly involved in the rate-limiting step of tyrosine transamination. In 2H2O solvent the reduction of the overall rates of transamination of both p-tyrosine and alpha-2H1-p-tyrosine occurred uncompetitively which suggests that the deuterium solvent effect is involved in the tautomerization of the external Schiff's base.