Primary Deuterium Isotope Effects Research Articles

Pathways in permanganate oxidation of mandelic acid: reactivity and selectivity of intermediate manganese species.

We report a comprehensive kinetic and product study of the oxidation of mandelic acid (MA) by permanganate in the pH range of 1-13, including a full account of total oxidizing equivalents (five and three-electron change in acidic and basic media, respectively). In the entire pH range, the reaction shows a primary kinetic deuterium isotope effect (kH/kD ≥8-9), indicating rate-limiting hydride transfer. The deuterium label in α-deutero-mandelic acid is retained in benzaldehyde. Benzaldehyde (BZ) is formed in post-rate limiting steps due to reactions involving manganese intermediates. In alkaline pH (≥13), in the presence of barium acetate, Mn(VI) is removed as insoluble blue barium manganate; the stoichiometry of the first step of reduction was found to be: MA + 2Mn(VII) → PGA + 2Mn(VI). Manganate, MnO42-, is directly reduced to MnO2 giving an additional mole of phenylglyoxylic acid (PGA). The experimentally observed ratio of benzaldehyde to phenylglyoxylic (BZ/PGA) provides a basis for discrimination between mechanistic choices that include direct reduction of Mn(V) to Mn(III) (in an acidic medium), disproportionation to Mn(IV) and Mn(VI) or oxidation to Mn(VI) by a second mole of permanganate. Interestingly, at pH 4, a stoichiometric, soluble Mn(IV) is observed for the first time for hydroxy-acid oxidation, reminiscent of the Guyard reaction. Because of the widespread use of permanganate as an environmentally green oxidant, results from mandelic acid oxidation have implications for the remediation of dissolved organic matter (DOM) including hydrocarbons and nitroaromatics in waste and groundwater.

Photochemistry of the Stilbenes in Methanol. Trapping the Common Phantom Singlet State.

A comparative study of the photochemistry of cis- and trans-stilbene in methanol shows that both isomers undergo methanol photoaddition giving similar yields of α-methoxybibenzyl in competition with cis-trans photoisomerization. Methanol addition occurs primarily following torsional relaxation of the lowest excited singlet states of each isomer, 1 c* and 1 t*, to a common twisted singlet excited state intermediate, 1 p*, initially called the phantom singlet state. The addition is consistent with the zwitterionic character of 1 p*. Ether forms by direct 1,2-addition of CH3OH to the central carbon atoms and by 1,1-addition following rearrangement to 1-benzyl-1-phenylcarbene. Use of CD3OD and GC/MS (gas chromatographic/mass spectroscopic) analysis of the ether products revealed that the ratio of carbene/direct addition pathways is higher starting from cis-stilbene. We conclude that 1 p* formed from 1 c* is hotter than 1 p* formed from 1 t*. Surprisingly, except for favoring the carbene pathway, the use of higher energy photons (254 vs 313 nm) does not affect the overall ether quantum yield starting from cis-stilbene but significantly enhances both pathways starting from trans-stilbene. It appears that carbene formation and direct methanol addition to higher trans-stilbene excited state(s) compete with relaxation to S1. Substitution of D for the vinyl Hs of stilbene enhances the direct addition pathway more than 2-fold and strongly suppresses the carbene insertion pathway, revealing a large, kpcd0/ kped2 = 6.3, primary deuterium isotope effect in the carbene rearrangement. The 2-fold increase in the ether quantum yield is due primarily to a 2.75-fold increase in the lifetime of 1 p* on deuterium substitution of the vinyl hydrogens.

Contribution of K99 and D319 to substrate binding and catalysis in the saccharopine dehydrogenase reaction

Saccharopine dehydrogenase catalyzes the NAD-dependent oxidative deamination of saccharopine to l-lysine and α-ketoglutarate. Lysine 99 is within hydrogen-bond distance to the α-carboxylate of the lysine substrate and D319 is in the vicinity of the carboxamide side chain of NADH. Both are conserved and may be important to the overall reaction. Replacing K99 with M gives decreases of 110-, 80- and 20-fold in the V 2/ K m values for lysine, α-ketoglutarate and NADH, respectively. Deuterium isotope effects on V and V/ K Lys increase, while the solvent deuterium isotope effects decrease compared to the C205S mutant enzyme. Data for K99M suggest a decreased affinity for all reactants and a change in the partition ratio of the imine intermediate to favor hydrolysis. A change in the bound conformation of the imine and/or the distance of the imine carbon to C4 of the nicotinamide ring of NADH is also suggested. Changing D319 to A decreases V 2/ K NADH by 33-fold. Primary deuterium and solvent deuterium isotope effects decrease compared to C205S suggesting a non-isotope sensitive step has become slower. NADH binds to enzyme first, and sets the site for binding of lysine and α-ketoglutarate. The slower step is likely the conformational change generated upon binding of NADH.

The oxidation state of active site thiols determines activity of saccharopine dehydrogenase at low pH

Saccharopine dehydrogenase catalyzes the NAD-dependent conversion of saccharopine to generate l-lysine and α-ketoglutarate. A disulfide bond between cysteine 205 and cysteine 249, in the vicinity of the dinucleotide-binding site, is observed in structures of the apoenzyme, while a dithiol is observed in a structure with AMP bound, suggesting preferential binding of the dinucleotide to reduced enzyme. Mutation of C205 to S gave increased values of V/Et and V/KEt at pH 7 compared to wild type. Primary deuterium and solvent deuterium kinetic isotope effects suggest the catalytic pathway, which includes the hydride transfer and hydrolysis steps, contributes more to rate limitation in C205S, but the rates of the two steps relative to one another remain the same. There is a large increase in the rate constants V1/Et and V1/KNADEt at pH values below 7 compared to WT. Data indicate the low pH increase in activity results from a decreased sensitivity of the C205S mutant enzyme to the protonation state of an enzyme group with a pKa of about 7, likely responsible for a pH-dependent conformational change. Reduction of WT and C205S mutant enzymes with TCEP gives equal activities at pH 6, consistent with the increased activity observed for the C205S mutant enzyme.

On the chemical mechanism of succinic semialdehyde dehydrogenase (GabD1) from Mycobacterium tuberculosis

Succinic semialdehyde dehydrogenases (SSADHs) are ubiquitous enzymes that catalyze the NAD(P)+-coupled oxidation of succinic semialdehyde (SSA) to succinate, the last step of the γ-aminobutyrate shunt. Mycobacterium tuberculosis encodes two paralogous SSADHs (gabD1 and gabD2). Here, we describe the first mechanistic characterization of GabD1, using steady-state kinetics, pH-rate profiles, 1H NMR, and kinetic isotope effects. Our results confirmed SSA and NADP+ as substrates and demonstrated that a divalent metal, such as Mg2+, linearizes the time course. pH-rate studies failed to identify any ionizable groups with pKa between 5.5 and 10 involved in substrate binding or rate-limiting chemistry. Primary deuterium, solvent and multiple kinetic isotope effects revealed that nucleophilic addition to SSA is very fast, followed by a modestly rate-limiting hydride transfer and fast thioester hydrolysis. Proton inventory studies revealed that a single proton is associated with the solvent-sensitive rate-limiting step. Together, these results suggest that product dissociation and/or conformational changes linked to it are rate-limiting. Using structural information for the human homolog enzyme and 1H NMR, we further established that nucleophilic attack takes place at the Si face of SSA, generating a thiohemiacetal with S stereochemistry. Deuteride transfer to the Pro-R position in NADP+ generates the thioester intermediate and [4A-2H, 4B-1H] NADPH. A chemical mechanism based on these data and the structural information available is proposed.

Investigating Inner-Sphere Reorganization via Secondary Kinetic Isotope Effects in the C−H Cleavage Reaction Catalyzed by Soybean Lipoxygenase: Tunneling in the Substrate Backbone as Well as the Transferred Hydrogen

This work describes the application of NMR to the measurement of secondary deuterium (2° (2)H) and carbon-13 ((13)C) kinetic isotope effects (KIEs) at positions 9-13 within the substrate linoleic acid (LA) of soybean lipoxygenase-1. The KIEs have been measured using LA labeled with either protium (11,11-h2-LA) or deuterium (11,11-d2-LA) at the reactive C11 position, which has been previously shown to yield a primary deuterium isotope effect of ca. 80. The conditions of measurement yield the intrinsic 2° (2)H and (13)C KIEs on k(cat)/K(m) directly for 11,11-d2-LA, whereas the values for the 2° (2)H KIEs for 11,11-h2-LA are obtained after correction for a kinetic commitment. The pattern of the resulting 2° (2)H and (13)C isotope effects reveals values that lie far above those predicted from changes in local force constants. Additionally, many of the experimental values cannot be modeled by electronic effects, torsional strain, or the simple inclusion of a tunneling correction to the rate. Although previous studies have shown the importance of extensive tunneling for cleavage of the primary hydrogen at C11 of LA, the present findings can only be interpreted by extending the conclusion of nonclassical behavior to the secondary hydrogens and carbons that flank the position undergoing C-H bond cleavage. A quantum mechanical method introduced by Buhks et al. [J. Phys. Chem. 1981, 85, 3763] to model the inner-sphere reorganization that accompanies electron transfer has been shown to be able to reproduce the scale of the 2° (2)H KIEs.

Formation of β-Ruthenium-Substituted Enones from Propargyl Alcohols

The reaction of the dienone ruthenium dicarbonyl dimer {[2,5-Ph-3,4-Tol(η5-C4CO)]Ru(CO)2}2 (7) with propargyl alcohol at room temperature gave a high yield of β-ruthenium-enal (E)-[2,5-Ph-3,4-Tol(η5-C4COH)]Ru(CO)2(CH═CHCHO) (8E), which was characterized spectroscopically and by X-ray crystallography. Reaction of 7 with pent-2-yn-1-ol led to the kinetic formation of the E-isomer (E)-[2,5-Ph-3,4-Tol(η5-C4COH)]Ru(CO)2[C(CH2CH3)═CHCHO] (10E-Et), which isomerized to an equilibrium mixture of Z- and E-isomers upon heating. The intramolecular nature of the 1,2-hydrogen shifts involved in these reactions was established by the absence of crossover products in the reaction of 7 with a mixture of PhC≡CCH2OH and PhC≡CCD2OH. A primary deuterium isotope effect (kH/kD ≈ 11) was seen on the product-forming step in the reaction of 7 with PhC≡CCHDOH. The reaction of PhC≡CCH3 with 7 produced the alkyne complex [2,5-Ph2-3,4-Tol2(η5-C4COH)]Ru(CO)2(η2-PhC≡CCH3) (14). The key step in the mechanism of the reaction of 7 with pro...

Temperature-Dependent Transitions Between Normal and Inverse Isotope Effects Pertaining to the Interaction of H−H and C−H Bonds with Transition Metal Centers

Deuterium kinetic isotope effects (KIEs) serve as versatile tools to infer details about reaction mechanisms and the nature of transition states, while equilibrium isotope effects (EIEs) associated with the site preferences of hydrogen and deuterium enable researchers to study aspects of molecular structure. Researchers typically interpret primary deuterium isotope effects based on two simple guidelines: (i) the KIE for an elementary reaction is normal (k(H)/k(D) > 1) and (ii) the EIE is dictated by deuterium preferring to be located in the site corresponding to the highest frequency oscillator. In this Account, we evaluate the applicability of these rules to the interactions of H-H and C-H bonds with a transition metal center. Significantly, experimental and computational studies question the predictability of primary EIEs in these systems based on the notion that deuterium prefers to occupy the highest frequency oscillator. In particular, the EIEs for (i) formation of sigma-complexes by coordination of H-H and C-H bonds and (ii) oxidative addition of dihydrogen exhibit unusual temperature dependencies, such that the same system may demonstrate both normal (i.e., K(H)/K(D) > 1) and inverse (i.e., K(H)/K(D) < 1) values. The transition between a normal and inverse EIE indicates that these systems do not demonstrate the typical monotonic variation predicted by the van't Hoff relationship. Instead, the calculated EIEs in these systems are 0 at 0 K, increase to a value greater than 1, and then decrease to unity at infinite temperature. This unusual behavior may be rationalized by considering the individual factors that contribute to the EIE. Specifically, the EIE may be expressed in the form EIE = SYM x MMI x EXC x ZPE (where SYM is the symmetry factor, MMI is the mass-moment of inertia term, EXC is the excitation term, and ZPE is the zero-point energy term), and the distinctive temperature profile results from the inverse ZPE (enthalpy) and normal [SYM x MMI x EXC] (entropy) components opposing each other and having different temperature dependencies. At low temperatures, the ZPE component dominates and the EIE is inverse, while at high temperatures, the [SYM x MMI x EXC] component dominates and the EIE is normal. The inverse nature of the ZPE term is a consequence of the rotational and translational degrees of freedom of RH (R = H, CH(3)) becoming low-energy isotopically sensitive vibrations in the product, while the normal nature of the [SYM x MMI x EXC] component results from deuterium substitution having a larger impact on the moment of inertia of the smaller molecule.

The tert-butoxyl radical mediated hydrogen atom transfer reactions of the Parkinsonian proneurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and selected tertiary amines

Previous studies have shown that the hydrogen atom transfer (HAT) reactions of tert-butoxyl radical from the Parkinsonian proneurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) occur with low selectivity at the allylic and non-allylic α-C–H positions. In this paper, we report a more comprehensive regiochemical study on the reactivity of the tert-butoxyl radical as well as on the associated primary kinetic deuterium isotope effects for the various hydrogen atom abstractions of MPTP. In addition, the results of a computational study to estimate the various C–H bond dissociation energies of MPTP are presented. The results of the present study show the allylic/non-allylic selectivity is approximately 73:21. The behavior of the tert-butoxyl radical mediated oxidation of MPTP contrasts with this reaction as catalyzed by monoamine oxidase B (MAO-B) that occurs selectively at the allylic α-carbon. These observations lead to the conclusion that the tert-butoxyl radical is not a good chemical model for the MAO-B-catalyzed bioactivation of MPTP.

Evidence for a Catalytic Dyad in the Active Site of Homocitrate Synthase from Saccharomyces cerevisiae

Homocitrate synthase (acetyl-coenzyme A: 2-ketoglutarate C-transferase; E.C. 2.3.3.14) (HCS) catalyzes the condensation of acetyl-CoA (AcCoA) and alpha-ketoglutarate (alpha-KG) to give homocitrate and CoA. Although the structure of an HCS has not been solved, the structure of isopropylmalate synthase (IPMS), a homologue, has been solved (Koon, N., Squire, C. J., and Baker, E. N. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 8295-8300). Three active site residues in IPMS, Glu-218, His-379, and Tyr-410, were proposed as candidates for catalytic residues involved in deprotonation of the methyl group of AcCoA prior to the Claisen condensation to give homocitrylCoA. All three of the active site residues in IPMS are conserved in the HCS from Saccharomyces cerevisiae. Site-directed mutagenesis has been carried out to probe the role of the homologous residues, Glu-155, His-309, and Tyr-320, in the S. cerevisiae HCS. No detectable activity was observed for the H309A and H309N mutant enzyme, but a slight increase in activity was observed for H309A in the presence of 300 mM imidazole, which is still 1000-fold lower than that of wild type (wt). The E155Q and E155A mutant enzymes exhibited 1000-fold lower activity than wt. The activity of E155A, but not of E155Q, could be partially rescued by formate; a K act of 60 mM with a modest 4-fold maximum activation was observed. In the presence of formate, E155A gives k cat, K AcCoA, and K alpha-KG values of 0.0031 s (-1), 13 muM, and 39 microM, respectively, while a primary kinetic deuterium isotope effect of about 1.4 was obtained on V, with deuterium in the methyl of AcCoA. The pH dependence of k cat for E155A in the presence of formate gave a p K a of 7.9 for a group that must be protonated for optimum activity, similar to that observed for the wt enzyme. However, a partial change was observed on the acid side of the profile, compared to the all or none change observed for wt giving a p K a of about 6.7. The k cat for E155Q decreased at high pH, similar to the wt enzyme, but was pH independent at low pH. The Y320F mutant enzyme only lost 25-fold activity compared to that of the wt, giving k cat, K AcCoA, and K alpha-KG values of 0.039 s (-1), 33 microM, and 140 microM, respectively, and a primary kinetic deuterium isotope effect of 1.3 and 1.8 on V/ K AcCoA and V, respectively; the pH dependence of k cat was similar to that of the wt. These data, combined with a constant pH molecular dynamics simulation study, suggest that a catalytic dyad comprising Glu-155 and His-309 acts to deprotonate the methyl group of AcCoA, while Tyr320 is likely not directly involved in catalysis, but may aid in orienting the reactant and/or the catalytic dyad.

The Mycobacterium tuberculosis Rv2540c DNA sequence encodes a bifunctional chorismate synthase.

BackgroundThe emergence of multi- and extensively-drug resistant Mycobacterium tuberculosis strains has created an urgent need for new agents to treat tuberculosis (TB). The enzymes of shikimate pathway are attractive targets to the development of antitubercular agents because it is essential for M. tuberculosis and is absent from humans. Chorismate synthase (CS) is the seventh enzyme of this route and catalyzes the NADH- and FMN-dependent synthesis of chorismate, a precursor of aromatic amino acids, naphthoquinones, menaquinones, and mycobactins. Although the M. tuberculosis Rv2540c (aroF) sequence has been annotated to encode a chorismate synthase, there has been no report on its correct assignment and functional characterization of its protein product.ResultsIn the present work, we describe DNA amplification of aroF-encoded CS from M. tuberculosis (MtCS), molecular cloning, protein expression, and purification to homogeneity. N-terminal amino acid sequencing, mass spectrometry and gel filtration chromatography were employed to determine identity, subunit molecular weight and oligomeric state in solution of homogeneous recombinant MtCS. The bifunctionality of MtCS was determined by measurements of both chorismate synthase and NADH:FMN oxidoreductase activities. The flavin reductase activity was characterized, showing the existence of a complex between FMNox and MtCS. FMNox and NADH equilibrium binding was measured. Primary deuterium, solvent and multiple kinetic isotope effects are described and suggest distinct steps for hydride and proton transfers, with the former being more rate-limiting.ConclusionThis is the first report showing that a bacterial CS is bifunctional. Primary deuterium kinetic isotope effects show that C4-proS hydrogen is being transferred during the reduction of FMNox by NADH and that hydride transfer contributes significantly to the rate-limiting step of FMN reduction reaction. Solvent kinetic isotope effects and proton inventory results indicate that proton transfer from solvent partially limits the rate of FMN reduction and that a single proton transfer gives rise to the observed solvent isotope effect. Multiple isotope effects suggest a stepwise mechanism for the reduction of FMNox. The results on enzyme kinetics described here provide evidence for the mode of action of MtCS and should thus pave the way for the rational design of antitubercular agents.

Chemical Mechanism of Homoisocitrate Dehydrogenase from Saccharomyces cerevisiae

Homoisocitrate dehydrogenase (HIcDH, 3-carboxy-2-hydroxyadipate dehydrogenase) catalyzes the fourth reaction of the alpha-aminoadipate pathway for lysine biosynthesis, the conversion of homoisocitrate to alpha-ketoadipate using NAD as an oxidizing agent. A chemical mechanism for HIcDH is proposed on the basis of the pH dependence of kinetic parameters, dissociation constants for competitive inhibitors, and isotope effects. According to the pH-rate profiles, two enzyme groups act as acid-base catalysts in the reaction. A group with a p K a of approximately 6.5-7 acts as a general base accepting a proton as the beta-hydroxy acid is oxidized to the beta-keto acid, and this residue participates in all three of the chemical steps, acting to shuttle a proton between the C2 hydroxyl and itself. The second group acts as a general acid with a p K a of 9.5 and likely catalyzes the tautomerization step by donating a proton to the enol to give the final product. The general acid is observed in only the V pH-rate profile with homoisocitrate as a substrate, but not with isocitrate as a substrate, because the oxidative decarboxylation portion of the isocitrate reaction is limiting overall. With isocitrate as the substrate, the observed primary deuterium and (13)C isotope effects indicate that hydride transfer and decarboxylation steps contribute to rate limitation, and that the decarboxylation step is the more rate-limiting of the two. The multiple-substrate deuterium/ (13)C isotope effects suggest a stepwise mechanism with hydride transfer preceding decarboxylation. With homoisocitrate as the substrate, no primary deuterium isotope effect was observed, and a small (13)C kinetic isotope effect (1.0057) indicates that the decarboxylation step contributes only slightly to rate limitation. Thus, the chemical steps do not contribute significantly to rate limitation with the native substrate. On the basis of data from solvent deuterium kinetic isotope effects, viscosity effects, and multiple-solvent deuterium/ (13)C kinetic isotope effects, the proton transfer step(s) is slow and likely reflects a conformational change prior to catalysis.

Multiple Roles of Arginine 181 in Binding and Catalysis in the NAD-Malic Enzyme from Ascaris suum

The NAD-malic enzyme catalyzes the oxidative decarboxylation of l-malate. Structures of the enzyme indicate that arginine 181 (R181) is within hydrogen bonding distance of the 1-carboxylate of malate in the active site of the enzyme and interacts with the carboxamide side chain of the nicotinamide ring of NADH, but not with NAD+. Data suggested R181 might play a central role in binding and catalysis in malic enzyme, and it was thus changed to lysine and glutamine to probe its potential function. A nearly 100-fold increase in the Km for malate and a 30-fold increase in the Ki for oxalate, an analogue of the enolpyruvate intermediate, in the R181Q and R181K mutants are consistent with a role for R181 in binding substrates. The mutant enzymes also exhibit a >10-fold increase in KiNADH, but only a slight or no change in KNAD, consistent with rotation of the nicotinamide ring into the malate binding site upon reduction of NAD+ to NADH. The activity of the R181Q mutant can be rescued by ammonium ion likely by binding in the pocket vacated by the guanidinium group of R181. Results suggest 2 mol of ammonia bind per mole of active sites with a high-affinity KNH4 of 0.7 +/- 0.1 mM and a low-affinity KNH4 of approximately 420 mM. Occupancy of the high-affinity site, likely by NH4+, results in an increase in the affinity of malate, oxalate, and NADH (with no change in NAD affinity), consistent with the above-proposed roles for R181. The second molecule to bind is likely neutral NH3, and its binding increases V/Et approximately 20-fold. Primary deuterium and 13C isotope effects measured in the absence and presence of ammonium ion suggest R181Q predominantly affects the rate of the reaction by changing the rate of the precatalytic conformational change. The isotope effects do not change upon binding the second mole of ammonia in spite of the 20-fold increase in V/Et. Thus, the R181Q mutant enzyme exists as an equilibrium mixture between active and less active forms, and NH3 stabilizes the more active conformation of the enzyme.

Control of Ionizable Residues in the Catalytic Mechanism of Tryptophan Synthase from Salmonella typhimurium

The tryptophan synthase alpha2beta2 complex catalyzes the last two steps in the biosynthesis of l-tryptophan in bacteria, plants, and fungi, the conversion of indole-3-glycerol phosphate and l-serine to l-tryptophan, glyceraldehyde 3-phosphate, and water. The beta-subunit binds pyridoxal 5'-phosphate and catalyzes the beta-replacement reaction with serine and indole. Structural, spectral, and kinetic studies indicate that different monovalent cations stabilize the alternative enzyme conformations and equilibrium distribution of the internal, external, and alpha-aminoacrylate Schiff base. To improve our understanding of the role of monovalent cations, the pH dependence of steady-state and pre-steady-state kinetic parameters and primary kinetic deuterium isotope effects were measured in the presence of l-serine and [alpha-2H]-l-serine in the absence and presence of Na+, K+, and Cs+. For the interpretation of the data obtained in this study, it was necessary to re-interpret a number of results published previously. Overall, data suggest that the enzyme exists in two conformers that equilibrate slowly either in the absence of substrates and monovalent cations or in the presence of K+ or Cs+, whereas they equilibrate faster in the presence of Na+. The rate of interconversion of the conformers increases as a group on the enzyme with a pKa of approximately 8 becomes deprotonated. The pH dependence of deuterium isotope effects is suggestive of a mechanism in which a pH-dependent conformational change that closes the active site precedes the chemical steps, likely a result of formation of one or more salt bridges. As the pH increases, the reaction becomes more committed to proceed to products, which causes the deuterium isotope effect to decrease to a value of unity at high pH. The closure of the site is modulated by the different monovalent cations and is fastest in the presence of Na+, which exhibits the maximum isotope effect of 5.7 (likely the intrinsic effect) on V/Kserine, and slowest in the presence of Cs+, which exhibits the smallest isotope effect of approximately 1.5. The isotope effect on V, in all cases, indicates a contribution to rate limitation from steps in the second half of the reaction. Finally, in the presence of Na+, the steady-state isotope effect on V is greater than that on the pre-steady-state rate constant for decay of the external Schiff base, suggesting that the rate of conversion of the two conformers of the internal aldimine contributes to the pre-steady-state rate, but not the steady-state rate because the high serine concentration traps the enzyme in the active E-serine complex before it can decay to the less active form.

Intrinsic Deuterium Kinetic Isotope Effects in Glutamate Mutase Measured by an Intramolecular Competition Experiment

Kinetic isotope effects (KIEs) provide a powerful tool to interrogate transition states of both enzymic and non-enzymic reactions, provided that one can measure the intrinsic KIE on the chemical step of interest, that is, the isotope effect undiminished by other isotope-insensitive steps that may contribute to the overall rate of reaction. For small molecules reacting in solution the KIE measured usually represents the intrinsic value; however, for enzymes this is seldom the case. Here we report the first measurement of the intrinsic KIE in an adenosylcobalamin enzyme (AdoCbl, coenzyme B12) for hydrogen atom transfer from substrate to coenzyme, which is a key step in the mechanism of this class of enzymes. For the B12 enzyme glutamate mutase the intrinsic deuterium KIE for hydrogen transfer from the substrate, (2S,3S)-3-methylaspartate, to 5’-deoxyadensosine is 4.1. This value is well within the semiclassical limit for a deuterium isotope effect and is much smaller than the anomalously large KIEs previously measured in other B12 enzymes and non-enzymatic model reactions, which were attributed to extensive hydrogen tunneling. Glutamate mutase one is of a group of AdoCbl-dependent enzymes that catalyze unusual isomerization reactions that formally involve a 1,2 hydrogen atom migration and proceed through a mechanism involving carbon-based free radical intermediates (Scheme 1). Radicals are generated by homolysis of the reactive cobalt–carbon bond of the coenzyme to form cob(II)alamin, a cobalt(II) intermediate, and the 5’-deoxyadenosyl radical. The adenosyl radical then abstracts the migrating hydrogen from the substrate to form 5’-deoxyadenosine and the substrate radical. The substrate radical next undergoes rearrangement to give the product radical, which is then quenched by hydrogen transfer from 5’deoxyadenosine to give the product and regenerate the 5’deoxyadenosyl radical. Finally, recombination of the adenosyl radical and cob(II)alamin to reform the coenzyme completes the catalytic cycle. Our interest in the mechanisms by which enzymes generate free radicals, as exemplified by dependent glutamate mutase, led us to undertake an extensive set of KIE measurements to examine how hydrogen abstraction from the substrate and coenzyme homolysis are coupled together. KIE measurements using deuteriumand tritium-labeled substrates and coenzyme have proved especially informative probes of the key steps of Co C bond homolysis and hydrogen atom abstraction from substrate. Pre-steady-state measurements on a number of enzymes have shown that hydrogen abstraction is kinetically coupled to Co C bond homolysis, as evidenced by the appearance of a kinetic isotope effect on cobalt–carbon bond homolysis when the enzymes are reacted with deuterated substrates. This observation implies that the 5’-dA radical is a highenergy intermediate that only has a fleeting existence. Furthermore, the KIEs reported for several AdoCbl enzymes are extremely large (ranging from 10 to 50), which has generally been attributed to hydrogen tunneling. In particular, extensive hydrogen tunneling in methylmalonyl-CoA mutase has been deduced from the temperature dependence of the deuterium isotope effect on hydrogen transfer and Co C bond homolysis. The KIEs discussed above were all measured indirectly by UV/Vis stopped-flow spectroscopy, using the spectroscopic changes associated with Co C bond homolysis as a convenient reporter of the kinetics. Although we originally reported a very large primary deuterium isotope effect, suggestive of tunneling, for the formation of cob(II)alamin and 5’-dA upon reaction of hologlutamate mutase with deuterated substrates (kH/kD= 28 with glutamate and 35 with methylaspartate), [11]

A Simple Method To Determine Kinetic Deuterium Isotope Effects Provides Evidence that Proton Transfer to Carbon Proceeds over and Not through the Reaction Barrier

A Simple Method To Determine Kinetic Deuterium Isotope Effects Provides Evidence that Proton Transfer to Carbon Proceeds over and Not through the Reaction Barrier

Hydrogen Transfer to Ketones Catalyzed by Shvo's Ruthenium Hydride Complex: A Mechanistic Insight

The Shvo catalyst is one of the most prominent examples of a hydrogen-transfer catalyst successfully applied in a broad scope of hydrogen-transfer processes. The reaction takes place by transferring a hydride (bonded to the metal center) and a proton (bonded to a ligand) to a double bond. The reaction mechanism for the hydrogen-transfer process, however, is a matter of controversy. Experimental studies by means of primary deuterium isotope effects on the hydrogenation of ketones via the active reducing form of the Shvo catalyst by the Casey and Bäckvall groups concluded that carbonyl hydrogenation is concerted. Nevertheless, it is not clear whether the reaction goes through an outer-sphere mechanism (without substrate coordination) or through an inner-sphere mechanism (with substrate coordination). In the present work several inner- and outer-sphere mechanisms are explored by means of theoretical methods. Energy reaction profiles clearly support the outer-sphere mechanism. Theoretically combined KIEs also suggests the concerted outer-sphere mechanism for this reaction. Some interesting features on the behavior of the Ru complex were found during the mechanistic investigations.

Mechanistic and Structural Studies of H373Q Flavocytochrome b2: Effects of Mutating the Active Site Base,

His373 in flavocytochrome b2 has been proposed to act as an active site base during the oxidation of lactate to pyruvate, most likely by removing the lactate hydroxyl proton. The effects of mutating this residue to glutamine have been determined to provide further insight into its role. The kcat and kcat/Klactate values for the mutant protein are 3 to 4 orders of magnitude smaller than the wild-type values, consistent with a critical role for His373. Similar effects are seen when the mutation is incorporated into the isolated flavin domain of the enzyme, narrowing the effects to lactate oxidation rather than subsequent electron transfers. The decrease of 3500-fold in the rate constant for reduction of the enzyme-bound FMN by lactate confirms this part of the reaction as that most effected by the mutation. The primary deuterium and solvent kinetic isotope effects for the mutant enzyme are significantly smaller than the wild-type values, establishing that bond cleavage steps are less rate-limiting in H373Q flavocytochrome b2 than in the wild-type enzyme. The structure of the mutant enzyme with pyruvate bound, determined at 2.8 A, provides a rationale for these effects. The orientation of pyruvate in the active site is altered from that seen in the wild-type enzyme. In addition, the active site residues Arg289, Asp 292, and Leu 286 have altered positions in the mutant protein. The combination of an altered active site and the small kinetic isotope effects is consistent with the slowest step in turnover being a conformational change involving a conformation in which lactate is bound unproductively.

A Quantum Mechanics/Molecular Mechanics Study of the Catalytic Mechanism of the Thymidylate Synthase

A theoretical study of the molecular mechanism of the thymidylate synthase-catalyzed reaction has been carried out using hybrid quantum mechanics/molecular mechanics methods. We have examined all of the stationary points (reactants, intermediates, transition structures, and products) on the multidimensional potential energy surfaces for the multistep enzymatic process. The characterization of these relevant structures facilitates the gaining of insight into the role of the different residues in the active site. Furthermore, analysis of the full energy profile has revealed that the step corresponding to the reduction of the exocyclic methylene intermediate by hydride transfer from the 6S position of 5,6,7,8-tetrahydrofolate (H4folate), forming dTMP and 7,8-dihydrofolate (H2folate), is the rate-limiting step, in accordance with the experimental data. In this step, the hydride transfer and the scission of an overall conserved active site cysteine residue (Cys146 in Escherichia coli) take place in a concerted but very asynchronous way. These findings have also been tested with primary and secondary deuterium, tritium, and sulfur kinetic isotope effects, and the calculations have been compared to experimental data. Finally, the incorporation of high-level quantum mechanical corrections to the semiempirical AM1 Hamiltonian into our hybrid scheme has allowed us to obtain reasonable values of the energy barrier for the rate-limiting step. The resulting picture of the complete multistep enzyme mechanism that is obtained reveals several new features of substantial mechanistic interest.

A Mechanistic Dichotomy in Scandium Ion-Promoted Hydride Transfer of an NADH Analogue: Delicate Balance between One-Step Hydride-Transfer and Electron-Transfer Pathways

The rate constant (kH) of hydride transfer from an NADH analogue, 9,10-dihydro-10-methylacridine (AcrH2), to 1-(p-tolylsulfinyl)-2,5-benzoquinone (TolSQ) increases with increasing Sc(3+) concentration ([Sc(3+)]) to reach a constant value, when all TolSQ molecules form the TolSQ-Sc(3+) complex. When AcrH2 is replaced by the dideuterated compound (AcrD2), however, the rate constant (kD) increases linearly with an increase in ([Sc(3+)]) without exhibiting a saturation behavior. In such a case, the primary kinetic deuterium isotope effect (kH/kD) decreases with increasing ([Sc(3+)]). On the other hand, the rate constant of Sc(3+)-promoted electron transfer from tris(2-phenylpyridine)iridium [Ir(ppy)3]to TolSQ also increases linearly with increasing ([Sc(3+)]) at high concentrations of Sc(3+) due to formation of a 1:2 complex between TolSQ*- and Sc(3+), [TolSQ*--(Sc(3+)2], which was detected by ESR. The significant difference with regard to dependence of the rate constant of hydride transfer on ([Sc(3+)]) between AcrH2 and AcrD2 in comparison with that of Sc3+-promoted electron transfer indicates that the reaction pathway is changed from one-step hydride transfer from AcrH2 to the TolSQ-Sc3+ complex to Sc3+-promoted electron transfer from AcrD2 to the TolSQ-Sc3+ complex, followed by proton and electron transfer. Such a change between two reaction pathways, which are employed simultaneously, is also observed by simple changes of temperature and concentration of Sc3+.