Pre-steady-state measurement of intrinsic secondary tritium isotope effects associated with the homolysis of adenosylcobalamin and the formation of 5'-deoxyadensosine in glutamate mutase

Glutamate mutase is one of a group of adenosylcobalamin-dependent enzymes that catalyze a variety of reactions that proceed through organic radical intermediates generated by homolytic fission of coenzyme's unique cobalt-carbon bond. For all the enzymes that have been examined, the homolysis step is kinetically indistinguishable from abstraction of hydrogen from the substrate (or protein), implying that deoxyadenosyl radical is formed only as a fleeting intermediate. To examine how these two steps are coupled together, we have used pre-steady-state, rapid quench techniques to measure the alpha-secondary tritium isotope effect associated with the formation of 5'-deoxyadenosine when the enzyme is reacted with [5'-(3)H]-adenosylcobalamin and L-glutamate. Surprisingly, a large inverse equilibrium isotope effect of 0.72 +/- 0.04 was found for the overall reaction, indicating that the 5'-C-H bonds become significantly stiffer on going from adenosylcobalamin to 5'-deoxyadenosine, even though the 5'-carbon remains formally sp(3) hybridized. The kinetic isotope effect for the formation of 5'-deoxyadenosine was 0.76 +/- 0.02, which suggests a late transition state for the reaction.     

Evidence for coupled motion and hydrogen tunneling of the reaction catalyzed by glutamate mutase

Glutamate mutase is one of a group of adenosylcobalamin-dependent enzymes that catalyze unusual isomerizations that proceed through organic radical intermediates generated by homolytic fission of the coenzyme's unique cobalt-carbon bond. These enzymes are part of a larger family of enzymes that catalyze radical chemistry in which a key step is the abstraction of a hydrogen atom from an otherwise inert substrate. To gain insight into the mechanism of hydrogen transfer, we previously used pre-steady-state, rapid-quench techniques to measure the alpha-secondary tritium kinetic and equilibrium isotope effects associated with the formation of 5'-deoxyadenosine when glutamate mutase was reacted with [5'-(3)H]adenosylcobalamin and L-glutamate. We showed that both the kinetic and equilibrium isotope effects are large and inverse, 0.76 and 0.72, respectively. We have now repeated these measurements using glutamate deuterated in the position of hydrogen abstraction. The effect of introducing a primary deuterium kinetic isotope effect on the hydrogen transfer step is to reduce the magnitude of the secondary kinetic isotope effect to a value close to unity, 1.05 +/- 0.08, whereas the equilibrium isotope effect is unchanged. The significant reduction in the secondary kinetic isotope effect is consistent with motions of the 5'-hydrogen atoms being coupled in the transition state to the motion of the hydrogen undergoing transfer, in a reaction that involves a large degree of quantum tunneling.     

Radical mechanisms in adenosylcobalamin-dependent enzymes

Adenolsylcobalamin-dependent enzymes catalyze free radical mediated reactions of their substrates. Stereochemical methods have been used to establish the nature of the primary radical initiation step in ribonucleoside triphosphate reductase. Kinetic isotope effects have been used to establish a kinetic coupling between cobalt-carbon bond cleavage and hydrogen atom abstraction from the substrate. Isotope effects have also been used to identify rate-limiting steps with wild type and mutant forms of the enzymes and in model reactions to assess tunneling contributions to hydrogen atom transfer steps. Computational methods have been employed to explore the pathways for functional group migration in the radical pathways. Analogs of substrates and of adenosylcobalamin have been used to explore the fidelity of the enzyme active sites and the radical pathways.     

Nanosecond transient absorption spectroscopy of coenzyme B12. Quantum yields and spectral dynamics

Photolysis of adenosylcobalamin leads to homolytic cleavage, similar to many of the B12-dependent enzyme reactions. Therefore, we have used photolysis to study the structure and lability of the cobalt-carbon bond. The nanosecond quantum yield for adenosylcobalamin is 0.23 +/- 0.04, higher than reported previously. The acidified form of adenosylcobalamin, so called "base-off" B12, has a much lower quantum yield at 0.045 +/- 0.015, demonstrating an inverse correlation between cobalt-carbon bond strength and quantum yield. Investigation of the wavelength dependence of the quantum yield shows that there is a highly efficient transmission of energy from the corrin ring to the cobalt-carbon bond. A comparison of nanosecond transient and static spectra showed small spectral differences. Therefore, any spectral relaxation of a sterically distorted corrin ring may be detectable only at sub-nanosecond timescales. Spectral analysis also provides data on the kinetics of recombination. In the absence of enzyme, geminate rebinding must be substantial, since the rate of Co(II) and deoxyadenosyl radical recombination is near the diffusion controlled limit. Therefore, it is likely that the enzyme functions to pull the geminate partners apart, perhaps as suggested previously, through a conformational change. The importance of geminate recombination in the mechanism of homolytic cleavage is further supported by a comparison of our results with picosecond transient absorption studies.     

How a protein generates a catalytic radical from coenzyme B(12): X-ray structure of a diol-dehydratase-adeninylpentylcobalamin complex

BACKGROUND: Adenosylcobalamin (coenzyme B(12)) serves as a cofactor for enzymatic radical reactions. The adenosyl radical, a catalytic radical in these reactions, is formed by homolysis of the cobalt-carbon bond of the coenzyme, although the mechanism of cleavage of its organometallic bond remains unsolved. RESULTS: We determined the three-dimensional structures of diol dehydratase complexed with adeninylpentylcobalamin and with cyanocobalamin at 1.7 A and 1.9 A resolution, respectively, at cryogenic temperatures. In the adeninylpentylcobalamin complex, the adenine ring is bound parallel to the corrin ring as in the free form and methylmalonyl-CoA-mutase-bound coenzyme, but with the other side facing pyrrole ring C. All of its nitrogen atoms except for N(9) are hydrogen-bonded to mainchain amide oxygen and amide nitrogen atoms, a sidechain hydroxyl group, and a water molecule. As compared with the cyanocobalamin complex, the sidechain of Seralpha224 rotates by 120 degrees to hydrogen bond with N(3) of the adenine ring. CONCLUSIONS: The structure of the adenine-ring-binding site provides a molecular basis for the strict specificity of diol dehydratase for the coenzyme adenosyl group. The superimposition of the structure of the free coenzyme on that of enzyme-bound adeninylpentylcobalamin demonstrated that the tight enzyme-coenzyme interactions at both the cobalamin moiety and adenine ring of the adenosyl group would inevitably lead to cleavage of the cobalt-carbon bond. Rotation of the ribose moiety around the glycosidic linkage makes the 5'-carbon radical accessible to the hydrogen atom of the substrate to be abstracted.     

Thermodynamic and kinetic characterization of Co-C bond homolysis catalyzed by coenzyme B(12)-dependent methylmalonyl-CoA mutase

Methylmalonyl-CoA mutase is a member of the family of coenzyme B(12)-dependent isomerases and catalyzes the 1,2-rearrangement of methylmalonyl-CoA to succinyl-CoA. A common first step in the reactions catalyzed by coenzyme B(12)-dependent enzymes is cleavage of the cobalt-carbon bond of the cofactor, leading to radical-based rearrangement reactions. Comparison of the homolysis rate for the free and enzyme-bound cofactors reveals an enormous rate enhancement which is on the order of a trillion-fold. To address how this large rate acceleration is achieved, we have examined the kinetic and thermodynamic parameters associated with the homolysis reaction catalyzed by methylmalonyl-CoA mutase. Both the rate and the amount of cob(II)alamin formation have been analyzed as a function of temperature with the protiated substrate. These studies yield the following activation parameters for the homolytic reaction at 37 degrees C: DeltaH(f)() = 18.8 +/- 0.8 kcal/mol, DeltaS(f)() = 18.2 +/- 0.8 cal/(mol.K), and DeltaG(f)() = 13.1 +/- 0.6 kcal/mol. Our results reveal that the enzyme lowers the transition state barrier by 17 kcal/mol, corresponding to a rate acceleration of 0.9 x 10(12)-fold. Both entropic and enthalpic factors contribute to the observed rate acceleration, with the latter predominating. The substrate binding step is exothermic, with a DeltaG of -5.2 kcal/mol at 37 degrees C, and is favored by both entropic and enthalpic factors. We have employed the available kinetic and spectroscopic data to construct a qualitative free energy profile for the methylmalonyl-CoA mutase-catalyzed reaction.     

Theoretical evaluation of the hydrogen kinetic isotope effect on the first step of the methylmalonyl-CoA mutase reaction

We have calculated hydrogen kinetic isotope effects (KIEs) for the first step of the methylmalonyl-CoA mutase reaction, including multidimensional tunneling correction at the zero curvature (ZCT) level, and compared them with the experimental values. Both alternative mechanisms of this step, concerted and stepwise, can be accommodated. It turned out to be essential to include Arg207 hydrogen-bonded to the reactant in the mechanism predicting simultaneous breaking of the Co-C bond of AdoCbl and hydrogen atom transfer. The consequence of the stepwise mechanism is a much larger facilitation of the homolytic dissociation of the carbon-cobalt bond by the enzyme than currently appreciated; our results suggest lowering of the activation energy by about 23 kcal mol(-1). We have also shown that large hydrogen KIEs of tunneling origin do not necessarily break the Swain-Schaad equation. Furthermore, when this equation does not hold, the exponent may be smaller in the presence of tunneling than it is at the semi-classical limit, indicating that nonclassical behavior may be a more common phenomenon than expected.     

Magnetic field effects on coenzyme B12-dependent enzymes: Validation of ethanolamine ammonia lyase results and extension to human methylmalonyl CoA mutase

Enzymes with radical-pair intermediates have been considered as a likely target for purported magnetic field effects in humans. The bacterial enzyme ethanolamine ammonia lyase and the human enzyme methylmalonyl-CoA mutase catalyze coenzyme B₁₂-dependent rearrangement reactions. A common step in the mechanism of these two enzymes is postulated to be homolysis of the cobalt-carbon bond of the cofactor to generate a spin-correlated radical pair consisting of the 5′-deoxyadenosyl radical and cob(II)alamin [Ado^· Cbl(II)]. Thus, the reactions catalyzed by these enzymes are expected to be sensitive to an applied magnetic field according to the same principles that control radical pair chemical reactions. The magnetic field effect on ethanolamine ammonia lyase reported previously has been corroborated independently in one of the authors' laboratory. However, neither the human nor the bacterial mutase from Propionibacterium shermanii exhibits a magnetic field effect that could be greater than about 15%, considering the error limit imposed by the uncertainty of the coupled assay. Our studies suggest that putative magnetic field effects on physiological processes are not likely to be mediated by methylmalonyl-CoA mutase. Bioelectromagnetics 18:506–513, 1997. © 1997 Wiley-Liss, Inc.     

Isotope effects for deuterium transfer between substrate and coenzyme in adenosylcobalamin-dependent glutamate mutase

A key step in the mechanism of all adenosylcobalamin-dependent enzymes is the abstraction of a hydrogen atom from the substrate by a 5'-deoxyadenosyl radical generated by homolytic fission of the coenzyme cobalt-carbon bond. We have investigated the isotope effects associated with this process for glutamate mutase reacting with deuterated glutamate. The kinetics of deuterium incorporation into 5'-deoxyadenosine (5'-dA) during the reaction were followed by rapid chemical quench, using HPLC and electrospray mass spectrometry to analyze the 5'-dA formed. The kinetics of 5'-dA formation are biphasic, comprising a rapid phase k(app) = 37 +/- 3 s(-)(1) and a slower phase k(app) = 0.9 +/- 0.4 s(-)(1). The mass spectral data clearly show that the faster phase is associated with the formation of monodeuterated 5'-dA whereas the slower phase is associated with the incorporation of a second and then a third deuterium into 5'-dA. This observation implies that a large inverse equilibrium secondary isotope effect is associated with the formation of 5'-dA from adenosylcobalamin. The primary deuterium kinetic isotope effects on V and V/K for the formation of 5'-dA were determined from time-based and competition experiments. (D)V = 2.4 +/-0.4 whereas (D)(V/K) = 10 +/- 0.4, implying that an isotopically insensitive step is partially rate-determining. The additional data provided by these experiments cause us to revise our interpretation of earlier UV-visible stopped-flow kinetic measurements of AdoCbl homolysis obtained with deuterated substrates.     

Catalytic reaction profile for alcohol oxidation by galactose oxidase

Galactose oxidase is a remarkable enzyme containing a metalloradical redox cofactor capable of oxidizing a variety of primary alcohols during enzyme turnover. Recent studies using 1-O-methyl alpha-D-galactopyranoside have revealed an unusually large kinetic isotope effect (KIE) for oxidation of the alpha-deuterated alcohol (kH/kD = 22), demonstrating that cleavage of the 6,6'-di[2H]hydroxymethylene C-H bond is fully rate-limiting for oxidation of the canonical substrate. This step is believed to involve hydrogen atom transfer to the tyrosyl phenoxyl in a radical redox mechanism for catalysis [Whittaker, M. M., Ballou, D. P., and Whittaker, J. W. (1998) Biochemistry 37, 8426-8436]. In the work presented here, the enzyme's unusually broad substrate specificity has allowed us to extend these investigations to a homologous series of benzyl alcohol derivatives, in which remote (meta or para) substituents are used to systematically perturb the properties of the hydroxyl group undergoing oxidation. Quantitative structure-activity relationship (QSAR) correlations over the steady state rate data reveal a shift in the character of the transition state for substrate oxidation over this series, reflected in a change in the magnitude of the observed KIE for these reactions. The observed KIE values have been shown to obey a log-linear correlation over the substituent parameter, Hammett sigma. For the relatively difficult to oxidize nitro derivative, the KIE is large (kH/kD = 12.3), implying rate-limiting C-H bond cleavage for the oxidation reaction. This contribution becomes less important for more easily oxidized substrates (e.g., methoxy derivatives) where a much smaller KIE is observed (kH/kD = 3.6). Conversely, the solvent deuterium KIE is vanishingly small for 4-nitrobenzyl alcohol, but becomes significant for the 4-methoxy derivative (kH2O/kD2O = 1.2). These experiments have allowed us to develop a reaction profile for substrate oxidation by galactose oxidase, consisting of three components (hydroxylic proton transfer, electron transfer, and hydrogen atom transfer) comprising a single-step proton-coupled electron transfer mechanism. Each component exhibits a distinct substituent and isotope sensitivity, allowing them to be identified kinetically. The proton transfer component is unique in being sensitive to the isotopic character of the solvent (H2O or D2O), while hydrogen atom transfer (C-H bond cleavage) is independent of solvent composition but is sensitive to substrate labeling. In contrast, electron transfer processes will in general be less sensitive to isotopic substitution. Our results support a mechanism in which initial proton abstraction from a coordinated substrate activates the alcohol toward inner sphere electron transfer to the Cu(II) metal center in an unfavorable redox equilibrium, forming an alkoxy radical which undergoes hydrogen atom abstraction by the tyrosine-cysteine phenoxyl free radical ligand to form the product aldehyde.     

Chemical probing of RNA with the hydroxyl radical at single-atom resolution

While hydroxyl radical cleavage is widely used to map RNA tertiary structure, lack of mechanistic understanding of strand break formation limits the degree of structural insight that can be obtained from this experiment. Here, we determine how individual ribose hydrogens of sarcin/ricin loop RNA participate in strand cleavage. We find that substituting deuterium for hydrogen at a ribose 5′-carbon produces a kinetic isotope effect on cleavage; the major cleavage product is an RNA strand terminated by a 5′-aldehyde. We conclude that hydroxyl radical abstracts a 5′-hydrogen atom, leading to RNA strand cleavage. We used this approach to obtain structural information for a GUA base triple, a common tertiary structural feature of RNA. Cleavage at U exhibits a large 5′ deuterium kinetic isotope effect, a potential signature of a base triple. Others had noted a ribose-phosphate hydrogen bond involving the G 2′-OH and the U phosphate of the GUA triple, and suggested that this hydrogen bond contributes to backbone rigidity. Substituting deoxyguanosine for G, to eliminate this hydrogen bond, results in a substantial decrease in cleavage at G and U of the triple. We conclude that this hydrogen bond is a linchpin of backbone structure around the triple.     

A Revised Mechanism for Human Cyclooxygenase-2

The mechanism of ω-6 polyunsaturated fatty acid oxidation by wild-type cyclooxygenase 2 and the Y334F variant, lacking a conserved hydrogen bond to the catalytic tyrosyl radical/tyrosine, was examined for the first time under physiologically relevant conditions. The enzymes show apparent bimolecular rate constants and deuterium kinetic isotope effects that increase in proportion to co-substrate concentrations before converging to limiting values. The trends exclude multiple dioxygenase mechanisms as well as the proposal that initial hydrogen atom abstraction from the fatty acid is the first irreversible step in catalysis. Temperature dependent kinetic studies reinforce the novel finding that hydrogen transfer from the reduced catalytic tyrosine to a terminal peroxyl radical is the first irreversible step that controls regio- and stereospecific product formation.     

Energetics of interaction between the G-protein chaperone, MeaB, and B12-dependent methylmalonyl-CoA mutase

MeaB is an auxiliary protein that supports the function of the radical B(12)-dependent enzyme, methylmalonyl-CoA mutase, although its precise role is not understood. Mutations in the human homolog of MeaB, MMAA, lead to methylmalonic aciduria, an inborn error of metabolism that can be fatal. To obtain insights into the function of this recently discovered protein, we have characterized the entropic and enthalpic contributions to DeltaGdegree (assoc) for complexation of MeaB (in the presence and absence of nucleotides) with methylmalonyl-CoA mutase (in the presence and absence of cofactor). The dissociation constant for binding of methylmalonyl-CoA mutase and MeaB ranges from 34 +/- 4 to 524 +/- 66 nm, depending on the combination of nucleotide and mutase form. Holomutase binds MeaB 15-fold more tightly when the nonhydrolyzable GTP analog, GMPPNP, is bound versus GDP. In contrast, the apomutase binds MeaB with similar affinity in the presence of either nucleotide. Our studies reveal that a large structural rearrangement accompanies interaction between these proteins and buries between approximately 4000 and 8600A(2) of surface area, depending on the combination of ligands in the active sites of the two proteins. Furthermore, we demonstrate that MeaB binds GTP and GDP with similar affinity (K(d) of 7.3 +/- 1.9 and 6.2 +/- 0.7 microm, respectively at 20 degrees C) and has low intrinsic GTPase activity (approximately 0.04 min(-1) at 37 degrees C), which is stimulated approximately 100-fold by methylmalonyl-CoA mutase. These studies provide insights into the energetics of interaction between the radical enzyme methylmalonyl-CoA mutase and MeaB, which are discussed.     

Photolysis of ortho-nitrobenzylic derivatives: the importance of the leaving group

Quantum yields for the photoinduced release of seven different commonly used leaving groups (LGs) from the o-nitroveratryl protecting group were measured. It was found that these quantum yields depend strongly on the nature of the LGs. We show that the quantum efficiency with which the LGs are released correlates with the stabilization that these LGs provide to o-nitrobenzyl-type radicals because radical stabilizing groups weaken the C-H bond that is cleaved in the photoinduced hydrogen atom transfer step, and hence lower the barrier for this process. At the same time these substituents lower the endothermicity of the thermal hydrogen atom transfer and thus increase the barrier for the reverse process, thereby enhancing the part of the initially formed aci-nitro intermediates which undergo cyclization (which ultimately leads to LG release). Radical stabilization energies computed by DFT methods are thus a useful predictor of the relative efficiency with which LGs are photoreleased from o-nitrobenzyl protecting groups.     

Carbon and hydrogen isotopic fractionation during anaerobic biodegradation of benzene

Compound-specific isotope analysis has the potential to distinguish physical from biological attenuation processes in the subsurface. In this study, carbon and hydrogen isotopic fractionation effects during biodegradation of benzene under anaerobic conditions with different terminal-electron-accepting processes are reported for the first time. Different enrichment factors (epsilon ) for carbon (range of -1.9 to -3.6 per thousand ) and hydrogen (range of -29 to -79 per thousand ) fractionation were observed during biodegradation of benzene under nitrate-reducing, sulfate-reducing, and methanogenic conditions. These differences are not related to differences in initial biomass or in rates of biodegradation. Carbon isotopic enrichment factors for anaerobic benzene biodegradation in this study are comparable to those previously published for aerobic benzene biodegradation. In contrast, hydrogen enrichment factors determined for anaerobic benzene biodegradation are significantly larger than those previously published for benzene biodegradation under aerobic conditions. A fundamental difference in the previously proposed initial step of aerobic versus proposed anaerobic biodegradation pathways may account for these differences in hydrogen isotopic fractionation. Potentially, C-H bond breakage in the initial step of the anaerobic benzene biodegradation pathway may account for the large fractionation observed compared to that in aerobic benzene biodegradation. Despite some differences in reported enrichment factors between cultures with different terminal-electron-accepting processes, carbon and hydrogen isotope analysis has the potential to provide direct evidence of anaerobic biodegradation of benzene in the field.     

Carbon isotope effects on the decarboxylation of carboxylic acids. Comparison of the lactate oxidase reaction and the degradation of pyruvate by H2O2.

The isotope effect at C-1 on the H2O2-catalysed decarboxylation of pyruvate (used as a model reaction for the enzymic reaction) increases between pH 3 and 10 from 1.0007 +/- 0.0004 to 1.0283 +/- 0.0014 (25 degrees C). This result indicates a change in the rate-determining step from formation of the tetrahedral intermediate to decarboxylation of this intermediate. Practically no isotope fractionation at C-1 (1.0011 +/- 0.0002, pH 6.0, 25 degrees C) is found in the lactate oxidase-catalysed decarboxylation of lactate, which is indicative for the existence of an irreversible O2-dependent step prior to the enzyme-catalysed decarboxylation. In addition, the result provides further evidence that dissociation of pyruvate and H2O2 from the enzyme can be excluded. The isotope effect at C-2 of lactate in the enzymic reaction (1.0048 +/- 0.0004) is attributed to the hydrogen transfer step from lactate to the coenzyme.     

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.     

Mechanistic understanding of Pyrococcus horikoshii Dph2, a [4Fe-4S] enzyme required for diphthamide biosynthesis

Diphthamide, the target of diphtheria toxin, is a unique posttranslational modification on eukaryotic and archaeal translation elongation factor 2 (EF2). The proposed biosynthesis of diphthamide involves three steps and we have recently found that in Pyrococcus horikoshii (P. horikoshii), the first step uses an S-adenosyl-L-methionine (SAM)-dependent [4Fe-4S] enzyme, PhDph2, to catalyze the formation of a C-C bond. Crystal structure shows that PhDph2 is a homodimer and each monomer contains three conserved cysteine residues that can bind a [4Fe-4S] cluster. In the reduced state, the [4Fe-4S] cluster can provide one electron to reductively cleave the bound SAM molecule. However, different from classical radical SAM family of enzymes, biochemical evidence suggest that a 3-amino-3-carboxypropyl radical is generated in PhDph2. Here we present evidence supporting that the 3-amino-3-carboxypropyl radical does not undergo hydrogen abstraction reaction, which is observed for the deoxyadenosyl radical in classical radical SAM enzymes. Instead, the 3-amino-3-carboxypropyl radical is added to the imidazole ring in the pathway towards the formation of the product. Furthermore, our data suggest that the chemistry requires only one [4Fe-4S] cluster to be present in the PhDph2 dimer.     

Carbon and Hydrogen Isotopic Fractionation during Anaerobic Biodegradation of Benzene

Compound-specific isotope analysis has the potential to distinguish physical from biological attenuation processes in the subsurface. In this study, carbon and hydrogen isotopic fractionation effects during biodegradation of benzene under anaerobic conditions with different terminal-electron-accepting processes are reported for the first time. Different enrichment factors () for carbon (range of −1.9 to −3.6‰) and hydrogen (range of −29 to −79‰) fractionation were observed during biodegradation of benzene under nitrate-reducing, sulfate-reducing, and methanogenic conditions. These differences are not related to differences in initial biomass or in rates of biodegradation. Carbon isotopic enrichment factors for anaerobic benzene biodegradation in this study are comparable to those previously published for aerobic benzene biodegradation. In contrast, hydrogen enrichment factors determined for anaerobic benzene biodegradation are significantly larger than those previously published for benzene biodegradation under aerobic conditions. A fundamental difference in the previously proposed initial step of aerobic versus proposed anaerobic biodegradation pathways may account for these differences in hydrogen isotopic fractionation. Potentially, C-H bond breakage in the initial step of the anaerobic benzene biodegradation pathway may account for the large fractionation observed compared to that in aerobic benzene biodegradation. Despite some differences in reported enrichment factors between cultures with different terminal-electron-accepting processes, carbon and hydrogen isotope analysis has the potential to provide direct evidence of anaerobic biodegradation of benzene in the field.     

Evidence that both protium and deuterium undergo significant tunneling in the reaction catalyzed by bovine serum amine oxidase

The magnitudes of primary and secondary H/T and D/T kinetic isotope effects have been measured in the bovine serum amine oxidase catalyzed oxidation of benzylamine from 0 to 45 degrees C. Secondary H/T and D/T kinetic effects are small and in the range anticipated from equilibrium isotope effects; Arrhenius preexponential factors (AH/AT and AD/AT) determined from the temperature dependence of isotope effects also indicate semiclassical behavior. By contrast, primary H/T and D/T isotope effects, 35.2 +/- 0.8 and 3.07 +/- 0.07, respectively, at 25 degrees C, are larger than semiclassical values and give anomalously low preexponential factor ratios, AH/AT = 0.12 +/- 0.04 and AD/AT = 0.51 +/- 0.10. Stopped-flow studies indicate similar isotope effects on cofactor reduction as seen in the steady state, consistent with a single rate-limiting C-H bond cleavage step for Vmax/Km. The comparison of primary and secondary isotope effects allows us to rule out appreciable coupling between the primary and secondary hydrogens at C-1 of the substrate. From the properties of primary isotope effects, we conclude that both protium and deuterium undergo significant tunneling in the course of substrate oxidation. These findings represent the first example of quantum mechanical effects in an enzyme-catalyzed proton abstraction reaction.